Synthetic salicylihalamides, apicularens and derivatives thereof

ABSTRACT

The present invention provides compounds having improved stability over that of natural benzolactones, and a process for synthesizing these compounds. These compounds exhibit anti-cancer activity and inhibit V-ATPase activity.

FIELD OF THE INVENTION

[0001] The present invention relates to the organic synthesis ofchemical compounds and anti-cancer pharmaceuticals, particularlymacrocyclic lactones having anti-cancer activity and vacuolar ATPaseinhibitory activity and methods of synthesis and use of these compounds.

BACKGROUND OF THE INVENTION

[0002] A number of biological metabolites isolated from natural sourcessuch as sponges, tunicates, and bacteria have been found to haveanti-cancer activity. These metabolites are macrocyclic lactones such assalicylihalamide and lobatamide, which appear to represent a newmechanistic class based on their cytotoxicity profiles when compared toother compounds in NCI's standard agents database. Apicularen has alsobeen shown to be cytostatic against human cancer cell lines (Kunze etal. (1998)). Macrolides were previously known to have cytotoxic activityand some possess anti-fungal or anti-bacterial properties. Theanti-cancer activity of a class of macrolides called salicylihalamidewas discovered by Boyd et al., (1997) in a screen of the cytotoxicactivity of extracts from a family of sponges (species Haliclona). Twonovel macrolides, salicylihalamides A and B were purified from theextract and tested in the NCI 60-cell line human tumor screen. Uponscreening, these compounds demonstrated a mean-graph profile that wasunlike any of the known tumor profiles of known anti-tumor agents.Therefore, these compounds represent a new class of anti-tumor agents.These compounds were especially effective against human solid tumor celllines. Solid tumors are usually the most resistant to drugs.

[0003] Various other macrocyclic lactones have been identified as havinganti-tumor activity. Included are lobatamides (isolated from Aplidiumlobatum), apicularens (isolated from Chondromyces), and oximidines(isolated from Pseudomonas). Lobatamides contain a similar enamide sidechain and core structure to salicylihalamides. However, lobatamidescontain an oxime methyl ether structure at the end of the enamide sidechain. The NCI 60-cell line human tumor screen profile of thelobatamides correlated with the profile of salicylihalamide (McKee etal., (1998)).

[0004] Apicularen A causes a potent growth inhibition of human cancercell lines, the induction of an apoptotic-like cell death, and theformation of mitotic spindles with multiple spindle poles and clustersof bundled actin from the cytoskeleton (Kunze et al., 1998; Jansen etal., 2000).

[0005] The NCI 60-cell line human tumor screen is a measure of theeffectiveness of a compound for inhibiting or killing various humancancers. It is a set of 60 different cancer cell lines against whichchemical compounds can be tested against to determine if the compoundhas anti-cancer activity. Each compound has an individual “fingerprint”based on effectiveness in killing each of the 60 cancer cell lines.

[0006] F{acute over ({acute over (u)})}rstner et al. (U.S. Pat. No.5,936,100) has used ring closing metathesis as a step in the synthesisof macrocyclic lactones containing one or more polar functional groups.Macrocycles of ring sizes ≧12 are challenging to synthesize because theprecursors tend to oligomerize.

[0007] The naturally occurring structure of salicylihalamide is unstableunder certain conditions. Salicylihalamides decompose in CDCl₃, due tothe unstable side chain (Snider and Song (2000)). The present inventionprovides macrocyclic lactones which exhibit improved stability over thenatural compound. The present invention also includes a process for thesynthesis of these compounds which is particularly flexible for makingvarious compounds. The natural compound has not previously beensynthesized and its structure was misidentified when it was purifiedfrom marine sponges of the genus Haliclona. Boyd et al., inPCT/US98/15011 disclosed the structure of natural salicylihalamide witha negative rotation and assigned the absolute configuration as 12R, 13S,15R. This assignment was incorrect because the isomer with the 12R, 13S,15R absolute configuration has a positive rotation and does not haveanti-cancer activity, as proven by the inventor in the presentapplication. Only the isomer with the 12S, 13R, 15S absoluteconfiguration has a negative rotation and anti-cancer activity.

[0008] The present invention describes the first synthesis of (+)-and(−)-salicylihalamide A and assigns the absolute configuration of thenatural product with negative rotation as 12S, 13R, 15S. It a highlyefficient, trans-selective ring-closing olefin metathesis for theassembly of the benzolactone skeleton and has been readily adapted toobtain a variety of analogs.

SUMMARY OF THE INVENTION

[0009] The invention includes a method of synthesis of a broad class ofcyclic benzolactones with chemotherapeutic activity which exhibitincreased stability over natural benzolactones. Included in theinvention are the compounds, compositions containing the compounds,methods of synthesis, and methods of treatment.

[0010] An embodiment of the invention is a composition comprising acompound of the formula:

[0011] wherein E is

[0012] X═O, S, NR²; Y═CH₂, O, S, NR²;

[0013] Q=O, NH;

[0014] F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R³;

[0015] R¹═H, Me;

[0016] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹ aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle;R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and Z is a contiguous linker whosepresence completes an 11 to 15 membered ring. The linker can containheteroatoms and substituents.

[0017] A further embodiment of the invention are compositions comprisingcompounds of the formulas:

[0018] wherein E is

[0019] X═O, S, NR²

[0020] Y═CH₂, O, S, NR²

[0021] Q=O, NH

[0022] F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R³

[0023] R¹═H, Me

[0024] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycleR³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and R⁴═R¹, C(O)R³, SO₂R³, R²

[0025] Another embodiment of the invention are compositions comprisingcompounds of the formulas:

[0026] where F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R ³;

[0027] R¹═H, Me;

[0028] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle;R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and

[0029] R⁴═R¹, C(O)R³, SO₂R³, R²

[0030] An embodiment of the invention is a composition wherein the poundis selected from the group consisting of:

[0031] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0032] An embodiment of the invention is a composition wherein thecompound is of the formula:

[0033] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0034] An embodiment of the invention is a composition comprising acompound of the formula:

[0035] An embodiment of the invention is a composition comprising acompound of the formula:

[0036] An embodiment of the invention is a composition comprising acompound of the formula:

[0037] An embodiment of the invention is a composition comprising acompound of the formula:

[0038] An embodiment of the invention is a composition comprising acompound of the formula:

[0039] An embodiment of the invention is a composition comprising acompound of the formula:

[0040] A further embodiment of the invention is the followingcompositions comprising compounds of the formulas: (These compounds havebeen tested for growth inhibitory activities against several cell lines,including human melanoma cell line SK-MEL-5 [see Example 7; Table 3], aswell a inhibition against reconstituted purified Vacuolar (H⁺)-ATPasefrom bovine brain [see Example 7; Table 5]).

[0041] where R=Z,Z-hexadienyl; Z,E-hexadienyl; a straight chain alkylcomprising 5 to 8 carbons (e.g. —(CH2)5Me); a straight chain alcohol(e.g. —O(CH2)4Me); and a straight chain diol (e.g. —S(CH2)4Me);

[0042] where R=Bu; Ph;

[0043] where R=Z,Z-hexadienyl; Z,E-hexadienyl; and a straight chainalkyl comprising 5 to 8 carbons (e.g. —(CH2)5Me);

[0044] where R=a straight chain alkyl comprising 5 to 8 carbons, astraight chain alcohol, a straight chain diol, —CCBu, or —CCph.

[0045] Another embodiment of the invention is a composition wherein thecompound is selected from the group consisting of:

[0046] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0047] Another embodiment of the invention is a composition wherein thecompound is of the formula:

[0048] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0049] Yet another embodiment of the invention is a process forpreparing a salicylihalamide comprising the steps of: a) synthesis of abenzolactone core; b) synthesis of an enamide side chain; and c) andaddition of the side chain by addition of a dienyllithium (28) to thebenzolactone core.

[0050] Another embodiment of the invention is a process for preparing asalicylihalamide comprising the steps of: a) synthesis of a benzolactonecore; b) synthesis of a side chain; and c) and addition of the sidechain to the benzolactone core.

[0051] Still another embodiment of the invention is a process forpreparing an Apicularen comprising the steps of: a) synthesis of abenzolactone core: b) synthesis of an enamide side chain; and c) andaddition of the side chain by addition of a dienyllithium (28) to thebenzolactone core.

[0052] Another embodiment of the invention is a process for preparing anApicularen comprising the steps of: a) synthesis of a benzolactone core:b) synthesis of a side chain; and c) and addition of the side chain tothe benzolactone core.

[0053] Another embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0054] wherein E is

[0055] X═O, S, NR²; Y═CH₂, O, S, NR²;

[0056] Q=O, NH;

[0057] F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R³;

[0058] R¹═H, Me;

[0059] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle;R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and Z is a contiguous linker whosepresence completes an 11 to 15 membered ring. The linker can containheteroatoms and substituents.

[0060] A further embodiment of the invention are compositions comprisingcompounds of the formulas:

[0061] wherein E is

[0062] X═O, S, NR²;

[0063] Y═CH₂, OS, NR²;

[0064] Q=O, NH;

[0065] F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R³;

[0066] R¹═H, Me;

[0067] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycleR³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and

[0068] R⁴═R¹, C(O)R³, SO₂R³, R²

[0069] Another embodiment of the invention are compositions comprisingcompounds of the formulas:

[0070] where F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R² , NR²C(O)R³, NR²SO₂R³, R³;

[0071] R¹═H, Me;

[0072] R²═R¹, straight chain saturated alkyl, straight chain unsaturatedalkyl, branched chain alkyl, branched chain unsaturated alkyl,cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl,CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle;R³═R² or CR¹=CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and

[0073] R⁴═R¹, C(O)R³, SO₂R³, R²;

[0074] where R=Z,Z-hexadienyl; Z,E-hexadienyl; a straight chain alkylcomprising 5 to 8 carbons, a straight chain alcohol or a straight chaindiol.

[0075] where R=Bu; Ph;

[0076] where R=Z,Z-hexadienyl; Z,E-hexadienyl; and a straight chainalkyl comprising 5 to 8 carbons;

[0077] where R=a straight chain alkyl comprising 5 to 8 carbons (e.g.—(CH2)5Me), a straight chain alcohol (e.g. —O(CH2)4Me), a straight chaindiol (e.g. —S(CH2)4Me), —CCBu, or —CCph.

[0078] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula selectedfrom the group consisting of:

[0079] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0080] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0081] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0082] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0083] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0084] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0085] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0086] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula selectedfrom the group consisting of:

[0087] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0088] An embodiment of the invention is a method of treating orpreventing cancer, comprising the step of administering to a patient atherapeutically effective amount of a compound of the formula:

[0089] wherein R=straight chain saturated alkyl or straight chainunsaturated alkyl that is comprised of a chain of 5 to 8 carbons.

[0090] An embodiment of the invention is a method for treating cancercomprising the step of contacting a tumor cell within a subject with amacrocyclic lactone of the present invention under conditions permittingthe uptake of said macrocyclic lactone by said tumor cell. The tumorcell may be derived from a tissue selected from the group consisting ofbrain, lung, liver, spleen, kidney, lymph node, small intestine, blood,pancreas, colon, stomach, breast, endometrium, prostate, testicle,ovary, skin, head, and neck, esophagus, and bone marrow. In a furtherembodiment, the subject is human. In another embodiment, the macrocycliclactone is contained within a liposome. In yet another embodiment, themacrocyclic lactone is administered intratumorally, in the tumorvasculature, local to the tumor, regional to the tumor, or systemically.In a further embodiment, the method comprises administering a secondchemotherapuetic agent to said subject. In a further embodiment, thesecond chemotherapeutic agent may be cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, famesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexateor any analog or derivative variant of the foregoing. In anotherembodiment, the method further comprises administering radiation to saidsubject. In another embodiment, the radiation is delivered local to acancer site. In yet another embodiment, the radiation is whole bodyradiation. The radiation may be γ-rays, X-rays, accelerated protons,microwave radiation, UV radiation or the directed delivery ofradioisotopes to tumor cells. In another embodiment, the method furthercomprises administering an anticancer gene to said subject. In anembodiment of the invention, the anticancer gene is a tumor suppressor.In another embodiment of the invention, the anticancer gene is aninhibitor of apoptosis. In another embodiment of the invention, theanticancer gene is an oncogene antisense construct.

[0091] It is a further embodiment of the present invention to provide amethod for inhibiting vacuolar ATPase (V-ATPase) proton-pumpingactivity. The method comprises contacting V-ATPase with the compounds ofthe present invention in an amount sufficient to inhibit the ATPaseproton-pumping activity of the V-ATPase. Inhibition of the V-ATPaseproton pumping activity by the compounds of the present invention isuseful, inter alia, for the treatment and prevention of cancer andosteoporosis.

[0092] An embodiment of the invention is a method for altering thephenotype of a tumor cell comprising the step of contacting the cellwith a macrocyclic lactone of the present invention, under conditionspermitting the uptake of said macrocyclic lactone by said tumor cell.The tumor cell may be derived from a tissue selected from the groupconsisting of brain, lung, liver, spleen, kidney, lymph node, smallintestine, blood, pancreas, colon, stomach, breast, endometrium,prostate, testicle, ovary, skin, head, and neck, esophagus, and bonemarrow. In an embodiment of the invention, the phenotype is selectedfrom the group consisting of proliferation, migration, contactinhibition, soft agar growth, cell cycling, invasiveness, tumorigenesis,and metastatic potential. In another embodiment of the invention, themacrocyclic lactone may be contained within a liposome.

[0093] Another embodiment of the invention is a method of treating asubject with cancer comprising the step of administering to said subjecta macrocyclic lactone of the present invention under conditionspermitting the uptake of said macrocyclic lactone by said cancer cell.In an embodiment of the invention, the subject is human.

[0094] Another embodiment of the invention is a method of suppressinggrowth of a tumor cell comprising contacting said cell with amacrocyclic lactone of the present invention, under conditionspermitting the uptake of said macrocyclic lactone by said tumor cell. Inanother embodiment, the macrocyclic lactone is contained within aliposome.

[0095] Another embodiment of the invention is a method of regulatingcell growth and proliferation in normal and malignant cells, comprisingthe step of administering, to an individual in need of said treatment, atherapeutically effective amount of a compound of the present invention.

[0096] Another embodiment of the present invention is a method ofinhibiting growth of proliferating cells comprising the step ofadministering, to the proliferating cells, a therapeutically effectiveamount of a compound of the present invention.

[0097] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF SUMMARY OF THE DRAWINGS

[0098] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

[0099]FIG. 1. Natural Salicylihalamides

[0100]FIG. 2. Strategy for introduction of side chain

[0101]FIG. 3. System of side chain synthesis

[0102]FIG. 4. Two general approaches to the synthesis of thebenzolactone core of salicylihalamide.

[0103]FIG. 5. Two specific approaches to the synthesis of thebenzolactone core of salicylihalamide.

[0104]FIG. 6. Synthesis of benzylic bromide 34 and synthesis of benzoicacid derivative 32

[0105]FIG. 7. Synthesis of alkyne 36

[0106]FIG. 8. Synthesis of benzolactone 58

[0107]FIG. 9. Fully optimized synthesis of benzolactone 74

[0108]FIG. 10. Introduction of the side chain and final deprotection

[0109]FIG. 11. Synthesis of p-bromobenzoate derivative ofsalicylihalamide and x-ray structure of p-bromobenzoate derivative ofsalicylihalamide

[0110]FIG. 12. Synthesis bis-olefins 328 a,b

[0111]FIG. 13. Ring closing metathesis synthesis of benzolactones 329a,b and 330 a,b

[0112]FIG. 14. Synthesis of acylazide 338

[0113]FIG. 15. Synthesis of salicylihalamides 301 a/301 c and analogs343-346.

[0114]FIG. 16. Synthesis of salicylihalamde analogs 349-354

[0115]FIG. 17. Synthesis of octanoate 355

[0116]FIG. 18. Synthesis of a fully saturated salicylihalamidederivative 362

[0117]FIG. 19. Synthesis of bis- and mono-protected forms ofsalicylihalamide derivatives 365-367

[0118]FIG. 20. Synthesis of Apicularen benzolactone core

[0119]FIG. 21. Synthesis of apicularen A and analogs.

[0120]FIG. 22. Methods to build a library of synthetic salicylihalamidesfrom common intermediates

[0121]FIG. 23. Elaborations of side chain-modified congeners ofsalicylihalamide

[0122]FIG. 24. Synthesis of apicularen and analogs

[0123]FIG. 25. ¹H NMR spectra of 301 a/301 c

[0124]FIG. 26. ¹H NMR spectra of 87

[0125]FIG. 27. ¹H NMR spectra of C22-E isomer of 87

[0126]FIG. 28. ¹H NMR spectra of 334 b

[0127]FIG. 29. ¹H NMR spectra of 335 b

[0128]FIG. 30. ¹H NMR spectra of 336

[0129]FIG. 31. ¹H NMR spectra of 337

[0130]FIG. 32. ¹H NMR spectra of 343

[0131]FIG. 33. ¹H NMR spectra of 344

[0132]FIG. 34. ¹H NMR spectra of 346

[0133]FIG. 35. ¹H NMR spectra of 345

[0134]FIG. 36. ¹H NMR spectra of 351

[0135]FIG. 37. ¹H NMR spectra of 355

[0136]FIG. 38. ¹H NMR spectra of 356

[0137]FIG. 39. ¹H NMR spectra of 361

[0138]FIG. 40. ¹H NMR spectra of 362

DETAILED DESCRIPTION OF THE INVENTION

[0139] Natural products that elicit a specific and unique biologicalresponse in mammalian cells represent valuable tools to identify, study,and target possible new gene products. In this context, the recentisolation of salicylihalamides A and B from the marine sponge Haliclonasp. is noteworthy. Pattern-recognition analysis of their uniquedifferential NCI 60-cell mean-graph screening profiles suggests that thesalicylihalamides belong to a potentially new mechanistic class ofantitumor compounds. Since their discovery in 1997, an emerging class ofnovel active metabolites have been isolated that structurally relate tothe salicylihalamides by virtue of an unprecedented highly unsaturatedenamide appended on a macrocyclic benzolactone. These include themechanistically related lobatamides, the potent cytostatic apicularens,selective inhibitors of oncogene-transformed cells (oximidines), as wellas compounds that induce low density lipoprotein (LDL) receptor geneexpression. However, the natural forms of these compounds are unstableunder certain conditions. The opportunity to develop novel chemistry aswell as accessing variants with increased stability, prompted theinventor to undertake the total synthesis of a modified salicylihalamideand modified apicularens. Included is a flexible synthetic strategy,which can be readily adapted to gain access to structural variants, aswell as other members of this intriguing class of anti-tumor compounds.

[0140] The inventor has accomplished the first synthesis of both (=)-and (−)-salicylihalamide A. The synthesis utilizes a highly efficient,trans-selective ring-closing olefin metathesis for the assembly of thebenzolactone skeleton and can be readily adapted to obtain a variety ofanalogs. The inventor synthesized the compound of the structuredisclosed in Boyd et al., (PCT/US98/1501 1) and found that the opticalrotation of synthetic salicylihalamide A (87) was ([α]_(D)=+20, c ,MeOH). The inventor found that this form did not possess anti-canceractivity. The (+) form was still inactive at 20 μM whereas naturalsalicylihalamide ((−) form) is active at 10 nM when tested againstSK-MEL-28 cells. This cell line is one of the cell lines in the NCI 60cell line screen. The inventor then accomplished the first synthesis of(−)-salicylihalamide A. It had a negative optical rotation like that ofnatural salicylihalamide A [α]_(D)=−35, c 0.7, MeOH) (Erickson et al.,1997). Salicylihalamide A with an (−) optical rotation does possessanti-cancer activity as does natural salicylihalamide, whereassalicylihalamide with a (+) optical rotation does not havechemotherapeutic activity.

[0141] The inventor has determined the absolute configuration ofsynthetic salicylihalarmide (87), suggesting that the absoluteconfiguration of natural salicylihalamide A was misassigned and has tobe corrected to the one represented by structure 89. The ultimate prooffor this assignment came from a crystallographic analysis of ap-bromobenzoate derivative of salicylihalamide 90, prepared from 73 in amanner similar to the synthesis of 74. This structure is shown in FIG.11. Comparison between synthetic salicylihalamide and naturalsalicylihalamide show the compounds to be identical in all respects(NMR, mass spectroscopy, IR, TLC (2 solvent systems: 50% ethylacetate inhexanes and 5% methanol in dichloromethane), and HPLC retentiontimes(co-elute on a normal phase silica gel, 5 micron column under 2different solvent conditions: 3% isopropanol in hexanes and 7%isopropanol in hexanes)) except for their rotations. The synthesis ofthe absolute configuration has also been accomplished by the inventor.See Examples 7 and 8.

[0142] The present invention also provides for methods/processes ofsynthesis of the compounds of the present invention which include twoequally active routes for its assembly (FIG. 4). One route features anesterification/intramolecular olefin metathesis sequence (RCM) to formthe C9-C10 bond and offers the advantage of operational simplicitycombined with functional group tolerance (A1+B1 gives AB; Path A; FIG.4). Despite the robustness of the RCM in carbon-carbon bond formation,it can only be implemented successfully for the synthesis ofsalicylihalamides if concomitant sterocontrol for the desired E-isomercan be exerted. As detailed in full below (Examples 7 and 8), theinventor has identified a highly E-selective RCM avenue tosalicylihalamide A. An alternative route involves cross coupling of asterodefined E-alkenyl organometallic fragment B2 with a bensyl halideA2 and is envisioned to join the C8-C9 bond with control and maintenanceof olefin geometry (Path B; FIG. 4). Importantly, both strategiesconverge to a common alkyne precursor B, adding flexibility to thesynthesis. The symbol P in FIG. 4 represents a generic symbol for ahydroxy protecting group.

[0143] For example, the present invention provides for a process forpreparing an Apicularen comprising:

[0144] a) synthesis of an Apicularen benzolactone core; and

[0145] c) addition of a side chain to the Apicularen benzolactone core.

[0146] In addition, the present invention provides for a process forpreparing a salicylihalamide comprising:

[0147] a) synthesis of a salicylihalamide benzolactone core; and

[0148] b) addition of a side chain to the salicylihalamide benzolactonecore.

[0149] In a preferred embodiment, the process for preparing asalicylihalamide comprises:

[0150] (a) synthesizing the compounds of formula:

[0151] (b) producing from the compounds of step (a), via an ring-closingmetathesis, the compound of formula:

[0152] wherein P=a hydroxyl protecting group

[0153] The process may further comprise:

[0154] (i) modifying the compounds of step (a) as follows:

[0155] Wherein n=0, 1, 2, or 3 and m=1, 2, or 3; R=alkyl; andF=functionality as defined in claim 1; and

[0156] (ii) Producing from the compounds in step (a), as defined in step(b) of above, the compounds of formula:

[0157] In a further preferred embodiment, the process for preparing asalicylihalamide comprises:

[0158] (a) synthesizing the compound of formula:

[0159] wherein X═I, Br, Cl, OSO2Aryl; F=functionality as defined inclaim 1; and P=a hydroxyl protecting group;

[0160] (b) synthesizing the compound of formula:

[0161] (c) synthesizing from the compound of step (b), via ahydrometallation, the compound of formula:

[0162] wherein m=1, 2, or 3; R=alkyl; P=a hydroxyl protecting group andL_(n)M is a ligated metal center with M═B, Zn, Zr, Pd, Cu, Li, Sn; and

[0163] (d) producing from the compounds of step (a) and (c), viametal-catalyzed cross coupling, the compound of formula:

[0164] In addition to their use as anti-tumor agents, compounds of thepresent invention have potential for treatment of osteoporosis, acondition in which bone resorption is increased resulting in weakeningof bone. The compounds can be administered alone or in combination withother treatments for osteoporosis. Other treatments for osteoporosis mayinclude, but are not limited to calcium supplements, estrogenreplacement for women, and treatment with bisphosphonates or growthfactors. The pharmaceutical compositions containing the compounds of thepresent invention are administered in the manner as described herein.

[0165] Salicylihalamide A exhibits a unique differential cytotoxicityprofile in the NCI 60-cell line human tumor assay and represents a novelmechanistic class of antitumor compounds. However, the inherent labilityof the enamide side-chain has compromised the development ofsalicylihalamide-based probe reagents for biochemical analysis. Theinventor has synthesized stable functional substitutes of theside-chain. Lobatamides A-F and salicylihalamide A-B are active and gavesimilar “signatures” in the NCI cancer panel. The inventor realized thatthe nature of the constituent atoms of the side-chain terminus could bevaried (alkylidene vs. methyloxime). Also, the stereochemical nature ofthe side-chain is unimportant, indicating a non-specific contact, ifany, with a putative biological receptor. The inventor has “mutated” theenamide without compromising function. FIG. 22 outlines methods to builda small library of synthetic “salicylihalamides” from commonintermediates 105 (ent-73, FIG. 9) and 107.

[0166] Compounds represented by 106 (ent-74, FIG. 9) are accessible from105 via Mitsunobu esterification with a variety of carboxylic acidsfollowed by deprotection (BBr₃). Aldehyde 107 is the central handle forolefination chemistry (stabilized and non-stabilized yields) and N-acylhydrazone formation. Compounds 110-113 are representative examples of alarger group of compounds that could be made by varying the R-group inthe reagents. Variations on the theme include geranyl ester 115 andallyl esters 109.

[0167] This method of synthesis is being used to provide new anti-cancercompounds. See also Examples 7 and 8. The inventor will use a cell-basedcytotoxicity assay to establish structure-activity relationships.Initial hits will be sent to the National Cancer Institute forCOMPARE-analysis in the NCI 60-cell line screen. Internal screeningagainst a broad range of primary human lung cancer cell lines will becarried out. The compounds of the present invention have also beentested for their ability to inhibit the growth of tumor cells, includingSK-MEL-5, H1299, J2009, H358, J2058, H175 and H1264. See Table 3 andTable 4 and Example 7 below.

[0168] Compounds may also be tested for activity in the yeastSaccharomyces cervisiae. Yeast-based screening methods are fast andwould allow for the identification of salicylihalamide's target viagenetic approaches, but only if a yeast homologue exists. Moreover, thecomplete genome of Saccharomyces cervisiae is sequenced and gene-chipsare available as powerful tools to look at expression patterns inresponse to drugs. One of the traditional drawbacks lies in the factthat yeast is relatively impermeable to small-molecule drugs and becomesresistant by overproduction of multidrug resistance pumps. To alleviatepotential problems, Δerg6 mutant strains, displaying reduced multi-drugresistance and more permeability to drugs due to more fluid membranes,will be used.

[0169] The compounds of the present invention can inhibit V-ATPaseproton-pumping activity. The V-ATPase proton-pumping inhibitory activityof the compounds of the present invention may be tested as described inExample 7 below (Table 5). Therefore, the present invention provides amethod for inhibiting the proton-pumping activity of V-ATPase bycontacting V-ATPase, either in vitro or in vivo with the compounds ofthe present invention. Inhibition of the proton-pumping activity ofV-ATPase is useful, inter alia, for the treatment and prevention ofcancer and osteoporosis. Salicylihalamide A has been shown to inhibitV-ATPase activity in crude membrane preparations of mammalian V-ATPases.See Boyd, M. R. et al., J. Pharmacol. Exp. Ther. 297, 114-120 (2001);see also, PCT publication WO 00/51589 of Boyd et al. V-ATPase iscomposed of an ATPase domain and a proton translocating membraneouschannel. The present invention demonstrates, through the use of apurified V-ATPase assay, that the mode of inhibition is through theblocking of the proton-pumping activity (or proton translocation, alsoknown as pore blocking) of V-ATPase and not through its ATP hydrolysisactivity. The present invention also demonstrates that the compounds ofthe present invention bind to the trans-membraneous proton channeldomain of V-ATPase and not to the ATPase domain. See Crider, Xie andStone, J. Biol. Chem. 269:17379-17381 (1994) for the experimentalprocedure for binding. This was shown by incubating select compounds ofthe present invention with the domains independently and showing that70% of the channel activity was inhibited at 1 nM of the compound whilethere was virtually no interference with ATPase activity at the sameconcentration. See Example 7 and Table 5 below. See also Bill . Crider,Xie and Stone, J. Biol. Chem. 269:17379-17381 (1994).

[0170] Probe reagents for biochemical analysis may be developed bymodifying selected positions without compromising function. FIG. 23outlines some elaborations of side chain-modified congeners. The exactnature of this side-chain (designated as R in FIG. 23) is contingent onthe screening results. In addition, Example 7 below describes otherchain modifications.

[0171] It is possible to attach probes to, or substitute for, theside-chain. Biotinylation allows cellular localization studies, affinitychromatography and ligand-based gel blotting analysis to be carried outusing avidin conjugates. Treatment of alcohol 116 withN-(+)-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester will givebiotinylated compound 117. The corresponding probes forphotoaffinity-labeling studies, 118 (R¹═B or C) can be obtained viaacylation with 4-benzoylbenzoic acid N-hydroxysuccinimide ester or4-azidobenzoic acid N-hydroxysuccinimide ester, respectively. If thesematerials are inactivated by the proposed modifications, iodophenol 119may be used as the starting material for derivatizations emanating fromthe aromatic ring. lodophenol 119 can be prepared from 116 (I₂).Selectivity for the para-position is usually observed. Substituting achloramine T/Na²⁵I reagent combination for iodine is a method tosynthesize radiochemicals. Sonogashira coupling of iodophenol 119 withpropargyl alcohol provides a handle (121) for introducing biotin (→122)or photolabels (→123/124) using the same acylating agents as describedabove. Radiochemicals are accessible via tritiation (Lindlar, ³H₂).Alkyne libraries (120) can be prepared using Sonogashira cross-couplingchemistry.

[0172] The inventor designed a C12-carboxamide as a synthon for theC12-methyl substituent (FIG. 4). To synthesize salicylihalamide analogsand probes otherwise not accessible, the C13-OH in 125 (ent-64) will bemasked as trilsopropylsilylether 126, followed by reduction of thecarboxamide (LiEt₃BH). The resulting primary alcohol can be converted toa coupling partner having a latent C12-hydroxymethyl substituent (121).Care must be taken to introduce the base-sensitive o-NO₂-benzylether. Anitroveratrylether is also a viable alternative. Mitsunobu inversion(acid 32), treatment with Grubbs' ruthenium carbene complex 57 andoxidative deprotection (DDQ) will provide cyclized material 128. Inclose analogy to the manipulations described for 104, a suitableside-chain (with respect to biological activity), identified through thestudies described in FIG. 22, will be introduced (→129) followed byphotodeprotection of the nitrobenzylether (350 nm, MeOH). At this point,the hydroxymethyl substituent serves its purpose as a handle forobtaining probe reagents 131 (acylation with N-hydroxysuccinimide estersas above, followed by desylilation of 130). Finally, similar probescontaining an amide linkage (134) can be constructed from azide 132using similar chemistry.

[0173] The inventor has also succeeded in the first and only totalsynthesis of Apicularen A and a variety of analogs. The syntheticsequence is outlined in FIGS. 20 and 43. For a description, see Example4. The initial lactonization approach of the inventor required thenatural configuration at the C15 hydroxyl-bearing center (15S) in a1,3-syn relationship with C13. A reagent-controlled mismatchedallylation (double diastereodifferentiation) was used for its formation.The inventor knew from work on salicylihalamides that ortho-alkoxybenzoates behave as poor electrophiles in esterification (lactonization)chemistry. Utilizing an intramolecular Mitsunobu inversion as the keyring-forming step could solve both problems. The substrate for thisreaction would require an inverted C15 alcohol in a 1,3-antirelationship with respect to C13. The intrinsic facial bias ofβ-alkoxyaldehydes favors such a relationship in their addition products.Based on numerous precedents, aldehyde 98 would be expected to yieldanti-homoallyl alcohol 99 with good stereocontrol by achelation-controlled (TiCl₄) addition of allyltrimethylsilane.Alternatively, the corresponding addition of allenylzinc (prepared insitu from propargylbromide and Zn) would yield stereoselectivelyanti-homopropargyl alcohol 145. Basic hydrolysis of benzo[1,3]dioxinone99 will release the carboxylic acid and the C11 hydroxyl functionalitiessimultaneously. A few scenarios can be envisioned for the subsequentMitsunobu ring-closure (135→101/102). The accepted mechanism for theMitsunobu inversion involves activation of the alcohol to a leavinggroup (RCH₂OP⁺Ph₃). The epimeric diol mixture 135 a-b would be expectedto give intermediates 136 a and 136 b. Both intermediates could react atC15 (with inversion) to give epimeric benzolactones 101 and 102,respectively, but the possibility of a competitive cyclization involvinga C11 activated hydroxy group has to be considered. However, only theundesired epimeric intermediate 136 b would be expected toparticipate—136 a can not reach the TTS required for an intramolecularSN₂ displacement—resulting in the formation of bridged lactone 137.Saponification of this material ultimately leads to a net recycling ofundesired 135 b to 135 a. Truncated apicularen 101 will be manipulatedin multiple ways to generate the natural product as well as a variety ofside-chain modified analogs. For example, the ozone adduct ofsilyl-protected derivative 138 (O₃, −78° C.) will be decomposed toaldehyde 139 (Me₂S), alcohol 140 (NaBH₄) and carboxylic acid 141 (H₂O₂),respectively. Apicularen A (4a) will be accessed from aldehyde 139 via asequence similar to the one successfully employed for the synthesis ofsalicylihalamide A (FIG. 10). Furthermore, compounds 139-141 arefunctionally equivalent to their corresponding salicylihalamidecounterparts 104, 107 and 114 (FIG. 22), and will be processed insimilar ways to deliver side chain-modified apicularen mimics 142-144(apicularens of general structure 142 are accessible directly from 101by intermolecular olefin metathesis).

[0174] Finally, alkyne 146 will be prepared from 145 in a manner similarto the one outlined for 101. The terminal alkyne provides the idealhandle for a Sonogashira cross-coupling to prepare apicularen-basedanalogs 147. In addition, the derived stannane 148 a and/or vinyliodide148 b will extend the range of cross-coupling reactions to the synthesisof 149 (R=aryl, 1-alkenyl) and enynes 150. Catalytic hydrogenation ofcompounds containing a triple bond will provide Z-alkenes (e.g. 151),potentially radiolabeled (³H₂, Lindlar cat.).

[0175] Examples 7 and 8 below further describe a preferred embodiment ofthe present invention.

[0176] In the present invention, all structural modifications that areuseful as modifications to salicylihalamides are also useful asmodifications to apicularens and other macrocyclic lactones.

[0177] X-ray crystallography is the study of the molecular structure ofcrystalline compounds through X-ray DIFFRACTION techniques. When anX-ray beam bombards a crystal, the atomic structure of the crystalcauses the beam to scatter in a specific pattern. This phenomenon, knownas X-ray diffraction, occurs when the wavelength of the X rays and thedistances between atoms in the crystal are of similar magnitude. X-raycrystallography provides information on the positions of individualatoms in the crystal, the distances between atoms, the angles of theatomic bonds, and other features of molecular geometry. X-raycrystallography is also used to determine the structure of proteins,nucleic acids, and other substances, such as small molecules(http://www.encyclopedia.com/articles/14056.html (6/7/00)).

[0178] Mass spectrometers use the difference in mass-to-charge ratio(m/e) of ionized atoms or molecules to separate them from each other.Mass spectrometry is therefore useful for quantitation of atoms ormolecules and also for determining chemical and structural informationabout molecules. Molecules have distinctive fragmentation patterns thatprovide structural information to identify structural components. Thegeneral operation of a mass spectrometer is: create gas-phase ions,separate the ions in space or time based on their mass-to-charge ratio,and measure the quantity of ions of each mass-to-charge ratio(http://www.scimedia.com/chem-ed/ms/ms-intro.htm(6/7/00)).

[0179] The temperature used for synthesis is, except where stated to bedifferent, in a range from about −78° C. to about 125° C., preferably 0°C. to 90° C.

[0180] The term “salicylihalamide derivative” as used herein refers tothose structures having a benzolactone core of the same conformation assalicylihalamide. Examples are:

[0181] where R=a straight chain alkyl comprising 5 to 8 carbons (e.g.—(CH2)5Me), a straight chain alcohol (e.g. —O(CH2)4Me) or a straightchain diol (e.g. —S(CH2)4Me);

[0182] where R=Bu or Ph;

[0183] where R=Z,Z-hexadienyl, Z,E-hexadienyl or a straight chain alkylcomprising 5 to 8 carbons (e.g. —(CH2)5Me).

[0184] The term “apicularen derivative” as used herein refers to thosestructures having a benzolactone core of the same conformation asapicularen. Examples are:

[0185] where R=a straight chain alkyl comprising 5 to 8 carbons (e.g.—(CH2)5Me), a straight chain alcohol (e.g. —O(CH2)4Me) or a straightchain diol (e.g. —S(CH2)4Me);

[0186] The present invention also provides for physiologicalcompositions comprising the compounds of the present invention. Aqueousphysiological compositions of the present invention comprise aneffective amount of a macrocyclic lactone of the present invention orpharmaceutically acceptable salt thereof, dissolved and/or dispersed ina pharmaceutically acceptable carrier and/or aqueous medium.

[0187] The phrases “physiologically, pharmaceutically and/orpharmacologically acceptable” refer to molecular entities and/orcompositions that do not produce an adverse, allergic and/or otheruntoward reaction when administered to an animal.

[0188] As used herein, “physiologically and/or pharmaceuticallyacceptable carrier” includes any and/or all solvents, dispersion media,coatings, antibacterial and/or antifungal agents, isotonic and/orabsorption delaying agents and/or the like. The use of such media and/oragents for pharmaceutical active substances is well known in the art.Except insofar as any conventional media and/or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. For human administration, preparations shouldmeet sterility, pyrogenicity, general safety and/or purity standards asrequired by FDA Office of Biologics standards.

[0189] The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds may generally be formulated for parental administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, intralesional and/or even intraperitoneal routes. Thepreparation of aqueous compositions that contain a therapeuticallyeffective amount of the macrocyclic lactones of the invention orpharmaceutically acceptable salts thereof as an active component and/oringredient will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions and/or suspensions; solid formssuitable for using to prepare solutions and/or suspensions upon theaddition of a liquid prior to injection can also be prepared; and/or thepreparations can also be emulsified.

[0190] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions and/or dispersions; formulations includingsesame oil, peanut oil and/or aqueous propylene glycol; and/or sterilepowders for the extemporaneous preparation of sterile injectablesolutions and/or dispersions. In all cases the form must be sterileand/or must be fluid to the extent that easy syringability exists. Itmust be stable under the conditions of manufacture and/or storage and/ormust be preserved against the contaminating action of microorganisms,such as bacteria and/or fungi.

[0191] Solutions of the active compounds as free base and/orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxyproplcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and/ormixtures thereof and/or in oils. Under ordinary conditions of storageand/or use, these preparations contain a preservative to prevent thegrowth of microorganisms.

[0192] Macrocyclic lactones of the present invention can be formulatedinto a composition in a neutral and/or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts and/or which areformed with inorganic acids such as, for example, hydrochloric and/orphosphoric acids, and/or such organic acids as acetic, oxalic, tartaric,mandelic, and/or the like.

[0193] The carrier can also be a solvent and/or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and/or liquid polyethylene glycol, and/or the like),suitable mixtures thereof, and/or vegetable oils. The proper fluiditycan be maintained, for example, by the use of a coating, such aslecithin, by the maintenance of the required particle size in the caseof dispersion and/or by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and/or the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars and/or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

[0194] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and/or freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, and/or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small tumorarea.

[0195] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and/or in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and/or the like can also beemployed.

[0196] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and/orthe liquid diluent first rendered isotonic with sufficient saline and/orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and/or intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and/or either added to 1000 ml ofhypodermoclysis fluid and/or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and/or 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

[0197] The macrocyclic lactones of the present invention may beformulated within a therapeutic mixture to comprise about 0.0001 to 1.0milligrams, and/or about 0.001 to 0.1 milligrams, and/or about 0.1 to1.0 and/or even about 10 milligrams per dose and/or so. Multiple dosescan also be administered.

[0198] Various routes of administration are contemplated for varioustumor types. For practically any tumor, systemic delivery iscontemplated. This will prove especially important for attackingmicroscopic or metastatic cancer. Where discrete tumor mass may beidentified, a variety of direct, local and regional approaches may betaken. For example, the tumor may be directly injected with themacrocyclic lactone. A tumor bed may be treated prior to, during orafter resection. Following resection, one could deliver the macrocycliclactone by a catheter left in place following surgery. One may utilizethe tumor vasculature to introduce the macrocyclic lactone into thetumor by injecting a supporting vein or artery. A more distal bloodsupply route also may be utilized.

[0199] In addition to the compounds formulated for parenteraladministration, such as intravenous and/or intramuscular injection,other pharmaceutically acceptable forms include, e.g., tablets and/orother solids for oral administration; liposomal formulations; timerelease capsules; and/or any other form currently used, includingcremes.

[0200] One may also use nasal solutions and/or sprays, aerosols and/orinhalants in the present invention. Nasal solutions are usually aqueoussolutions designed to be administered to the nasal passages in dropsand/or sprays. Nasal solutions are prepared so that they are similar inmany respects to nasal secretions, so that normal ciliary action ismaintained. Thus, the aqueous nasal solutions usually are isotonicand/or slightly buffered to maintain a pH of 5.5 to 6.5. In addition,antimicrobial preservatives, similar to those used in ophthalmicpreparations, and/or appropriate drug stabilizers, if required, may beincluded in the formulation. Various commercial nasal preparations areknown and/or include, for example, antibiotics and/or antihistaminesand/or are used for asthma prophylaxis.

[0201] Additional formulations which are suitable for other modes ofadministration include vaginal suppositories and/or pessaries. A rectalpessary and/or suppository may also be used. Suppositories are soliddosage forms of various weights and/or shapes, usually medicated, forinsertion into the rectum, vagina and/or the urethra. After insertion,suppositories soften, melt and/or dissolve in the cavity fluids. Ingeneral, for suppositories, traditional binders and/or carriers mayinclude, for example, polyalkylene glycols and/or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%.

[0202] Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand/or the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulationsand/or powders. In certain defined embodiments, oral pharmaceuticalcompositions will comprise an inert diluent and/or assimilable ediblecarrier, and/or they may be enclosed in hard and/or soft shell gelatincapsule, and/or they may be compressed into tablets, and/or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand/or used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and/or the like. Suchcompositions and/or preparations should contain at least 0.1% of activecompound. The percentage of the compositions and/or preparations may, ofcourse, be varied and/or may conveniently be between about 2 to about75% of the weight of the unit, and/or preferably between 25-60%. Theamount of active compounds in such therapeutically useful compositionsis such that a suitable dosage will be obtained.

[0203] The tablets, troches, pills, capsules and/or the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,and/or gelatin; excipients, such as dicalcium phosphate; adisintegrating agent, such as corn starch, potato starch, alginic acidand/or the like; a lubricant, such as magnesium stearate; and/or asweetening agent, such as sucrose, lactose and/or saccharin may be addedand/or a flavoring agent, such as peppermint, oil of wintergreen, and/orcherry flavoring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carrier.Various other materials may be present as coatings and/or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, and/or capsules may be coated with shellac, sugar and/or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and/or propylparabens as preservatives, a dye and/orflavoring, such as cherry and/or orange flavor.

[0204] In certain embodiments of the present invention, the use of lipidformulations and/or nanocapsules is contemplated for the introduction ofthe macrocyclic lactones of the present invention or pharmaceuticallyacceptable salts thereof into host cells. Lipid formulations andnonocapsules may be prepared by methods well known in the art.

[0205] “Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.

[0206] Liposomes within the scope of the present invention can beprepared in accordance with known laboratory techniques. In onepreferred embodiment, liposomes are prepared by mixing liposomal lipids,in a solvent in a container, e.g., a glass, pear-shaped flask. Thecontainer should have a volume ten-times greater than the volume of theexpected suspension of liposomes. Using a rotary evaporator, the solventis removed at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

[0207] In the alternative, liposomes can be prepared in accordance withother known laboratory procedures: the method of Bangham et al. (1965),the contents of which are incorporated herein by reference; the methodof Gregoriadis, as described in DRUG CARRIERS IN BIOLOGY AND MEDICINE,G. Gregoriadis ed. (1979) pp. 287-341, the contents of which areincorporated herein by reference; the method of Deamer and Uster (1983),the contents of which are incorporated by reference; and thereverse-phase evaporation method as described by Szoka andPapahadjopoulos (1978). The aforementioned methods differ in theirrespective abilities to entrap aqueous material and their respectiveaqueous space-to-lipid ratios.

[0208] A physiological composition comprising the liposomes will usuallyinclude a sterile, pharmaceutically acceptable carrier or diluent, suchas water or saline solution.

[0209] The present invention also provides kits comprising themacrocyclic lactones of the present invention or pharmaceuticallyacceptable salts thereof. Such kits will generally contain, in suitablecontainer means, a pharmaceutically acceptable formulation of themacrocyclic lactones of the present invention in a pharmaceuticallyacceptable formulation.

[0210] The container means will generally include at least one vial,test tube, flask, bottle, syringe and/or other container means, intowhich the macrocyclic lactones of the present invention formulation areplaced, preferably, suitably allocated. The kits may also comprise asecond container means for containing a sterile, pharmaceuticallyacceptable buffer and/or other diluent.

[0211] The kits of the present invention will also typically include ameans for containing the vials in close confinement for commercial sale,such as, e.g., injection and/or blow-molded plastic containers intowhich the desired vials are retained.

[0212] Irrespective of the number and/or type of containers, the kits ofthe invention may also comprise, and/or be packaged with, an instrumentfor assisting with the injection/administration and/or placement of theultimate the macrocyclic lactones of the present invention orpharmaceutically acceptable salts thereof within the body of an animal.Such an instrument may be a syringe, pipette, forceps, and/or any suchmedically approved delivery vehicle.

[0213] In order to increase the effectiveness of the macrocycliclactones of the present invention, it may be desirable to combine thesecompositions with other agents effective in the treatment ofhyperproliferative disease, such as anti-cancer agents. An “anti-cancer”agent is capable of negatively affecting cancer in a subject, forexample, by killing cancer cells, inducing apoptosis in cancer cells,reducing the growth rate of cancer cells, reducing the incidence ornumber of metastases, reducing tumor size, inhibiting tumor growth,reducing the blood supply to a tumor or cancer cells, promoting animmune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with themacrocylic lactones and other agent(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the same ordifferent time, wherein one composition includes the macrocyclic lactoneand the other includes the second agent(s).

[0214] Cancer therapies may include a variety of combination therapieswith both chemical and radiation based treatments. Combinationchemotherapies may include, for example, macrocylic lactones, cisplatin(CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, gemcitabien, navelbine, famesyl-proteintansferase inhibitors, transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate, or any analog or derivative variant of theforegoing.

[0215] The compounds may also be used together with immunotherapy.Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

[0216] In yet another embodiment, the compounds of the present inventionmay be combined with gene therapy in which a therapeutic polynucleotideis administered before, after, or at the same time as the macrocycliclactone of the present invention. Delivery of a vector encoding one ofthe following gene products will have a combined anti-hyperproliferativeeffect on target tissues. In the following sections, genes which can beused in gene therapy in conjunction with administration of themacrocyclic lactones will be described. For example, the compounds maybe administered together with an expression construct comprising a tumorsuppressor gene, such as, but not limited to, the p53 and p16 gene.

[0217] Other genes that may be employed according to the presentinvention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras,myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genesinvolved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF,or their receptors) and MCC, the Bcl-2 protein family genes, andICE-like protease genes.

[0218] Furthermore, the compouns of the present invention may be used incombination with surgery.

[0219] It is contemplated that other agents may be used in combinationwith the present invention to improve the therapeutic efficacy oftreatment. These additional agents include immunomodulatory agents,agents that affect the upregulation of cell surface receptors and GAPjunctions, cytostatic and differentiation agents, inhibitors of celladehesion, or agents that increasre the sensitivity of thehyperproliferative cells to apoptotic inducers. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate thechemotherapeutic abililties of the present invention by establishment ofan autocrine or paracenne effect on hyperproliferative cells. Increasesintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with the presentinvention to improve the anti-hyerproliferative efficacy of thetreatments. Inhibitors of cell adhesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastin. It is furthercontemplated that other agents that increase the sensitivity of ahyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

[0220] Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

EXAMPLES

[0221] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 1 Installation of the Enamide Side Chain

[0222] The macrocyclic salicylates of the invention may be decoratedwith one of two classes of an unsaturated side chain connected via acommon enamide linkage. This acid and base sensitive construct wasintroduced at a late stage in the synthesis (FIG. 1). The inventor feltthat the addition of an alkenyllithium 20 a or 20 b to a stereodefinedE- or Z-alkenyl isocyanate 19 would offer the distinct advantage of mildreaction conditions and control of stereochemistry. Isocyanate 19 wasderived from the corresponding α,β-unsaturated carboxylic acid (acylazide formation/Curtius rearrangement), in turn accessible from a C17aldehyde 22 via Horner-Wadsworth-Emmons homologation. FIG. 4 shows anexample of side chain synthesis.

[0223] The required alkenyllithium fragment for the synthesis of bothsalicylihalamides and apicularens was derived from the corresponding 1Z,3Z-hexadienyl halide via transmetalation with t-butyllithium. Beginningwith a Sonagashira coupling of Z-1-bromobutene (23) withtrimethylsilylacetylene, enyne 24 was obtained in 93% yield. Exchange ofthe silyl group with iodine or bromine (NIS or NBS) was accomplished inthe presence of AgNO₃. Although bromide 26 was isolated as a pureZ-compound, the corresponding iodide 25 had succumbed to partialisomerization of the double bond (Z:E=4:1). This ratio was reversedduring the subsequent reduction (i. Cy₂BH, ii. HOAc) of the triple bondin 25 whereas only partial isomerization had occurred during thecorresponding reduction of bromide 26 (28, Z:E=4:1). The subsequenttransmetalation/isocyanate addition not only proceeded without furtherisomerizations, but effected the crucial key-transformation to obtainN-(E-styryl)-heptadienamide 30 in high yields.

Example 2 Salicylihalamides: Exploration of Fragment Synthesis andCoupling Protocols

[0224] It was then necessary to synthesize the benzolactone core of thesalicylihalamides. A connectivity analysis pointed to two equallyattractive routes for its assembly (31, FIG. 4). The first one featuredan esterification/intramolecular olefin metathesis sequence to form theC9-C10 bond and offered the advantage of operational simplicity combinedwith functional group tolerance (Path A). However, the efficiency aswell as stereoselectivity (E vs Z) for medium to large ring-formingmetatheses are substrate dependent. Alternatively, a Pd(0)-catalyzedcross coupling of a stereodefined E-alkenyl organometallic fragment 35with benzylic bromide 34 joined the C8-C9 bond with maintenance ofolefin geometry (Path B). Importantly, both strategies converged to acommon alkyne precursor 36, adding flexibility to the synthesis. Thereis versatility in the available options for synthesizing fragment 36 buta particular C12-C13 aldol bond construction was singled out from theoutset for the following reasons: (1) absolute and relativestereochemistry can be controlled by the use of camphorsultamauxiliaries, and (2) a functionalized C12-substituent (carboxylate orhydroxymethyl) may prove useful as an additional handle from which todevelop molecular probes for mode-of-action studies.

[0225] Although related to salicylic acid, fragments 32 and 34 are notbe easily obtained from a salicylic acid precursor. To access benzylicbromide, a de novo synthesis from non-aromatic precursors was performed.Indeed, by stirring a mixture of 1-methoxy-1,3-cyclohexadiene (39) withethyl propiolate (40) at 170° C., a Diels-Alder reaction takes place(41), which is followed by a pericyclic extrusion of ethylene yieldingbenzoate 42. A radical bromination completes the synthesis of benzylicbromide 34 in 50% yield for the two step sequence. On the other hand,derivative 32 was obtained from 2,6-dihydroxybenzoic acid (43). Afterpreparation of the known aryl triflate 44, a Stille coupling withallyltributylstannane introduced the ortho-allyl substituent in 95%yield. Treatment of this material with the magnesium salt of allylalcohol induced a transesterification with concomitant release ofacetone (→45). Finally, protection of the phenol and palladium-catalyzeddeprotection of the allyl ester provided benzoic acid derivative 32.This sequence is critical to avoid conjugation of the double bond.

[0226] The synthesis of alkyne 36 starts from readily available aldehyde47, prepared in two steps from 1,3-propanediol (i. NaH, THF,t-BuMe₂SiCl; ii. (COCl)₂, DMSO, NEt₃, CH₂Cl₂, −78° C.). Aldol reactionof aldehyde 47 with the borylenolate derived from2-N-acetylbornanesultam 46 gave a separable mixture of two aldolproducts, 48 and its epimer, in an 85:15 ratio. An X-raycrystallographic analysis of 48 revealed its absolute configuration.Aldehyde 37, obtained via a one step reduction of protected aldolderivative 49, was subjected to a stereoselective syn-aldol reactionwith the titanium enolate derived from (2)-N-(4-pentynoyl) bornanesultam38. Only a single diastereomer could be detected by ¹H NMR-analysis ofthe crude reaction mixture. Protection followed by reduction (LiEt₃BH)of the N-acyl sulfonamide 51 delivered primary alcohol 52. Tosylateformation and another reduction (LiEt₃BH) completed the synthesis offragment 36 (78%, 2 steps).

[0227] The assembly of the alkyne 36 with the aryl sector was thenachieved (FIG. 8). In one of two approaches, this involved ahydrometallation of the triple bond, followed by cross coupling withbenzylic bromide 34 (path B, FIG. 4). In this context, zirconocene 35 a(ML_(n)=ClCp₂Zr) was considered the most appealing nucleophilic couplingpartner. Stereodefined 1-(E)-alkenylzirconocenes are readily accessiblevia a functional group tolerant hydrozirconation and engage incross-coupling chemistry with a variety of benzyl halides. The use of insitu prepared C1HZrCp₂ (LiEt₃BH, Cl₂ZrCp₂) was critical for obtainingreproducible results. As demonstrated in Table 1, these conditionsproved to be general for a variety of alkyne/benzyl halide combinationsincluding the cross coupling with benzyl bromide 34 (entry 6), properlysubstituted for the synthesis of salicylihalamides. Attempts to obtainzirconocene 35 a via hydrozirconation of alkyne 36 were unsuccessful(FIG. 8). In contrast, alkyne 36 did undergo smooth hydrostannylation(Bu₃SnH, AIBN, toluene) as a prelude to an alternative Stille couplingwith benzylic bromide 34. The corresponding vinylstannane 35 c wasextremely prone to protodestannylation, yielding terminal alkene 54 uponworkup. The desired coupling product 53 could be obtained in a singlecase via Suzuki cross-coupling in 15% yield. Again, the culprit seemedto be an inefficient hydrometallation as starting alkyne 36 accountedfor the remaining mass balance. It is not known why this particularalkyne resists hydrozirconation or hydroboration. Possibly, theparticular combination of protecting groups and stereochemistry of 36could render the alkyne sterically inaccessible to the bulkyhydrometallating agents. Clearly, salicylihalamides and variants mightstill be accessible using this procedure if modified C9-C17 alkynefragments prove to be better substrates for hydrometallation chemistry.

[0228] A ring-closing olefin metathesis- path to salicylihalamides wasexplored. The p-methoxybenzyl protecting group of alkene 54 wasoxidatively deprotected (DDQ). The inventor experienced difficulties inthe attempts to acylate the resulting alcohol 55 with an acylating agentderived from benzoic acid derivative 32. The C15 epimer of bis-olefin 56could not be obtained. This was suspected to be due to increasedelectron density (o-MeO-substituent) at the electrophilic carbonylcenter. Indeed, a reactivity umpolung provided by a Mitsunobuesterification (carboxylic acid 32 acting as the nucleophile) wasessential and delivered bis-olefin 56 in over 90% yield. Ironically,this material is useless for the synthesis of salicylihalamides due tothe inverted configuration at C15 (a consequence of the Mitsunobuinversion). The inventor tested the ability of Grubbs' ruthenium carbenecatalyst 57 to effect an intramolecular ring-closing olefin metathesis.Benzolactone 58 was closed in essentially quantitative yield using thisapproach. A separable mixture of isomers resulted with the Z-isomerpredominating (3:1). This is in contrast to the excellent E-selectivityobserved in the correctly configured diastereomeric series reminiscentof the natural product (vide infra). TABLE 1 Pd-Ctalyzed cross-couplingof vinylzirconocenes with benzylic halides Entry Alkyne ArCH₂X ProductYld (%)  1  2

BnCl BnBr

65 68  3 p-MeO-BnBr

78  4 o-Br-BnBr

82  5 o-Br, p-F-BnBr

72  6 34

63  7

BnBr

65  8

o-Br-BnBr

72  9 10

BnCl BnBr

73 75 11 12

BnCl BnBr

71 67 13 14

BnCl BnBr

72 76 15

BnBr

53 16

BnCl

73

Example 3 Salicylihalamides: Total Synthesis of Enantiomer with PositiveRotation (Structure Proposed by Boyd et al. in PCT/US98/15011

[0229] A procedure delivering gram quantities of benzolactone 75 ispresented in FIG. 8. The inventor opted for an enantioselectiveallylation of aldehyde 59 to set absolute stereochemistry at C15. Thecorresponding homoallyl alcohol 60, obtained in 96% yield, was protectedfollowed by oxidative double bond-cleavage (72% yield, 3 steps).Treatment of the corresponding aldehyde 62 with the in situ preparedZ(O)-titanium enolate derived from (2R)-N-(4-pentenoyl)bomanesultam 63produced exclusively one diastereomeric aldol product 64 in 95% yield.Protection (→65), carboxamide reduction (→68) and fluoride-assistedliberation of the C15 alcohol (→69) proceeded with high overall yields(61%, 5 steps). Treatment of alcohol 69 with carboxylic acid 32 underMitsunobu conditions (EtO₂CN═NCO₂Et, PPh₃) yielded bis-olefin 70 (98%),setting the stage for the key intramolecular ring-closing olefinmetathesis. In contrast to bis-olefin 56 (vide supra), exposure of 70 toa catalytic amount of Grubbs' ruthenium carbene complex 57preferentially produced the desired E-isomer 72 with selectivity of9-10:1 (E:Z). Metathesis of the closely related bis-olefin 71 to thecorresponding benzolactone 73 under identical conditions was far lessselective (E/Z=3:1). The inventor has achieved an extremely efficientsynthesis of the lactone core of salicylihalamides, delivering gramquantities of primary alcohols 74 or 75 (obtained from 72/73 byoxidative deprotection with DDQ) in 35% overall yield from aldehyde 59(13 steps). Derivative 74, designed as a side-chain analog ofsalicylihalamide (1 a), provided crystals suitable for X-ray diffractionstudies, confirming the assigned structure and stereochemistry (FIG.11b). Interestingly, the solid-state conformation of the macrolactone 74differs significantly from the more bowl-like topography found in thesolution structure of salicylihalamide A.

[0230] All that remained to complete the total synthesis was theintroduction of the side-chain followed by final deprotection. Alcohol75 was oxidized to aldehyde 76 with Dess-Martin periodinane (DMP) in 93%yield (FIG. 10). Moving forward, aldehyde 76 had to be homologated toα,β-unsaturated carboxylic acid 77. This was achieved with excellenttrans-selectivity by a Homer-Wadsworth-Emmons homologation withtrimethylsilyl dimethylphosphonoacetate followed by a slightly acidicaqueous workup. Following acylazide formation (diphenylphosphoryl azide,NEt₃) and Curtius rearrangement (benzene, 80° C.), isocyanate 80 wasobtained as a chromatographically pure compound. The dienamideside-chain was then installed. Incorporation proceeded smoothly via theaddition of hexadienyllithium (27, t-BuLi; see FIG. 2) to a cold (−78°C.) solution of isocyanate 80. All attempts to remove the protectinggroups uniformly met with failure, leading to decomposition. Tocircumvent this problem, the inventor used a protecting groupinterconversion at an earlier stage. Carboxylic acid 77 and t-butylester78 were not viable options, yielding intractable mixtures ofunidentified materials under the reaction conditions required to removethe protecting groups (BBr₃, CH₂Cl₂, −78° C.). In contrast, BBr₃ cleanlyremoved both methyl and MOM-ether protecting groups of methylester 79(obtained from 76 as an inseparable mixture of E/Z-isomers), after whichthe corresponding E- and Z-methylesters 82 and 83 could be separated.Note that 83 is a useful intermediate for the synthesis ofsalicylihalamide B (1 b). Hydrolysis (Ba(OH)₂.8H₂O) of the majortrans-methylester 82 was followed by complete silylation (TBSCI, DMF,imidazole). Upon workup, silylester hydrolysis had occurred and thecorresponding acid 84 was converted to 86 via addition ofZ,Z-dienyllithium (28, t-BuLi) to isocyanate 85. The total synthesis wascompleted by deprotection of the silylether protecting groups with a 1:1complex of HF-pyridine in THF, affording synthetic salicylihalamide A(1) in x% yield. This material was identical in all respects (¹H and ¹³CNMR, IR, UV, MS) to natural salicylihalamide A. However, the opticalrotation ([α]_(D)=+20, c , MeOH) of synthetic salicylihalamide A (87)was of opposite sign to the one recorded for natural salicylihalamide A([α]_(D)=−35, c 0.7, MeOH).

[0231] The absolute configuration of natural salicylihalamide A (1 a)was originally misassigned in Boyd et al., 1997. The correctconfiguration is represented by structure 89. The ultimate proof forthis assignment came from a crystallographic analysis of ap-bromobenzoate derivative of salicylihalamide 90, prepared from 73 in amanner similar to the synthesis of 74. The structure is shown in FIG.11(a). The inventor synthesized (+)-salicylihalamide A and found that itdid not have chemotherapeutic activity. (−)-salicylihalamide A does havechemotherapeutic activity. The (+) form was still inactive at 20 μMwhereas natural salicylihalamide ((−) form) is active at 10 nM whentested against SK-MEL-28 cells. This cell line is one of the cell linesin the NCI 60 cell line screen.

[0232] The inventor has accomplished the first synthesis ofsalicylihalamide A. The approach features a highly efficient,trans-selective ring-closing olefin metathesis for the assembly of thebenzolactone skeleton and installation of a dienamide side-chain.

Example 4 Apicularens: Synthesis of the Macrocyclic Fragment

[0233] The inventor has synthesized apicularen A (4 a) by the processshown in FIG. 20. Dihydropyranone 94 was considered a usefulintermediate from which to build the tetrahydropyranyl ring present inapicularens. The most straightforward approach for its assemblyconstitutes an enantioselective hetero-Diels-Alder reaction of aldehyde92 with Danishefsky's diene (1-methoxy-3-(trimethylsilyloxy)butadiene).The ease of catalyst synthesis and high enantioselectivities observedwas the reason for using chromium complex 93 for the hetero-Diels-Alderreaction of Danishefsky's diene with aldehyde 92, obtained via oxidativecleavage of alkene 91 (an intermediate of the salicylihalamidesynthesis). Dihydropyranone 94 was obtained in 60% yield after treatmentof the Diels-Alder adducts with trifluoroacetic acid. The enantiomericexcess was determined by chiral HPLC-analysis (CHIRALCEL® OD-H; 84% ee).Copper(I)-catalyzed conjugate addition of vinylmagnesium bromide to thismaterial gave exclusively the 2,6-trans substituted tetrahydropyranone95. The next step involved stereoselective ketone reduction and avariety of agents are known that selectively deliver hydride from thepseudoaxial (e.g. NaBH₄ or SmI₂) or pseudoequatorial (L-SELECTRIDE®)position. None of these conditions were successful however, and aninseparable epimeric mixture of alcohols 96 was obtained in all cases.The corresponding ketone most likely exists as an equilibrium mixture oftwo equally populated conformers. Such a situation would provide anepimeric mixture if both conformers react with comparable rates, evenwith highly selective reducing agents. This problem could be solved bydelaying the reduction until after the macrocyclization provided thatthis will reduce the conformational flexibility of thetetrahydropyranone ring. In the meantime, the epimeric mixture wassilylated followed by hydroboration (BH₃, THF; H₂O₂) of alkene 97 andoxidation of the resulting primary alcohol with TPAP(tetrapropylammonium perruthenate). Completion of the macrocyclicportion of the apicularens entailed an allylation/lactonizationsequence. A low selectivity was observed during the reaction of aldehyde98 with Brown's allyldiisopinocampheylborane, delivering a 77: 23mixture of diastereomeric homoallyl alcohols 99 and 100 (65%). This wasnot completely unexpected given the intrinsic facial bias of β-alkoxyaldehydes for 1,3-anti addition products. Moreover, 8% of enantiomericaldehyde 98 was present and would be expected to give productenantiomeric to 99, further lowering the syn/anti selectivity butincreasing the enantiomeric purity of 100. Treatment of the desiredmajor diastereomer 100 (mixture of C11 epimers) with NaH effected thecrucial lactonization in 70% yield. After silylether removal, epimeric(C11) alcohols 101 and 102 could be separated by chromatography. Thechemical shift values and coupling constants of protons H3 through H15(400 MHz NMR) of 101 are nearly identical to the values reported forapicularen A.

Example 5 Apicularens: Total Synthesis of Apicularen A and Analogs

[0234] The transformation of compounds 101 and 102 [which correspond to224 and 225 of FIG. 25, respectively] into apicularen A and a variety ofanalogs is shown in FIG. 25 and follows the general procedures disclosedfor the installation of the side chains of salicylihalamide A andanalogs (see FIGS. 13-15 and Examples 7 and 8). TABLE 2 ¹H NMR data ofapicularen A and macrocycle 101 [D₆]acetone apicularen A 101 H δ(m, J inHz) δ(m, J in Hz) 8a 3.34 (dd, 9.7/14.8) 3.34 (dd, 9.6/15.2) 8b 2.44(dd, 1.8/14.8) 2.45 (dd, 1.6/15.2) 9 3.87 (dddd, 1.7/4.8/8.2/9.7) 3.88(dddd, 1.2/4.8/8.0/10.0) 10a 1.93 (ddd, 4.2/4.8/12.8) 1.93 (ddd,4.8/4.8/12.8) 10b 1.48 (ddd, 8.5/8.8/12.8) 1.49 (ddd, 8.4/8.4/12.8) 113.98 (dddd, 4.1/5.1/7.6/8.8) 3.99 (ddddd, 4.0/4.1/5.1/7.6/8.8) 12a 1.68(ddd, 5.1/7.1/12.8) 1.68 (ddd, 5.2/7.2/12.8) 12b 1.52 (ddd,4.8/7.6/12.8) 1.52 (ddd, 4.4/7.2/12.8) 13 4.25 (dddd, 2.2/4.8/7.1/10.8)4.26 (dddd, 2.0/5.0/7.0/10.8) 14a 1.83 (ddd, 10.8/10.9/14.7) 1.83 (ddd,10.8/10.8/14.4) 14b 1.57 (ddd, 2.2/2.3/14.7) 1.58 (ddd, 2.0/2.4/14.4) 155.42 (dddd, 2.3/6.3/6.3/10.9) 5.48 (dddd, 2.4/5.6/5.6/10.0)

Example 6 Anti-Cancer Activity of Synthetic Salicylihalamides

[0235] The anti-cancer activity of the synthesized (+)- and(−)-salicylihalamide-A compounds of the present invention (compounds 78in FIG. 10 and 301 a/301 c in FIG. 14.) was indistinguishable. Incontrast, sythetic (+)-salicylihalamide A (compound 87 in FIG. 9), thecompound with the absolute configuration proposed by Boyd et al., wsacompleterly inactive in the same NCI 60-cell line screen.Salicylihalamide derivatives, showing increased stability over naturalsalicylihalamide, were tested in the NCI 60 cell-line screen. Thisscreen is an art recognized model. Prior to assay, synthesized compoundswere stored in dimethylsulfoxide at −70° C. Five different testconcentrations of each compound were prepared by diluting the stocksolution into complete medium. Sample concentrations were 2× the finalconcentration and range from 10⁻⁶ and 10⁻¹⁰ molar. An aliquot of eachconcentration of each compound was added to separate microtiter wellscontaining the respective cell lines in culture medium. The plates wereincubated for 48 hours at 37° C. with 5% CO₂ and 100% humidity. Adherentcells were fixed by addition of cold 50% trichloracetic acid andincubation at 4° C. for 60 minutes. Suspended cells were fixed to thebottom of the well by addition of 80% trichloracetic acid. Thesupernatant was removed from each well and then each well was washedwith deionized water and allowed to dry. Sulforhodamine B solution wasadded to each well and allowed to incubate for 10 minutes at roomtemperature. Unbound sulforhodamine B was removed by washing with 1%acetic acid. The wells were allowed to dry. Bound sulforhodamine B wassolubilized using 10 mM Tris base and the optical density at 515nanometers was determined. The synthesized salicylihalamide derivativesexhibited anti-cancer activity profiles.

[0236] A 1:1 mixture of salicylihalamide A (1) and C22-E isomer 211^(3,5d-g) was indistinguishable, within the limits of experimentalerror, from natural salicylihalamide A based on comparative testing inthe National Cancer Institute 60-Cell Screen.

Example 7 Overall Synthesis Scheme of Synthetic Salicylihalamides

[0237] Compounds 328 a and 328 b (FIG. 11) were prepared as describedfor the corresponding enantiomers 70 and 71 (FIG. 8) by usingenantiomeric starting materials. Thus the enantiomers of the lpc2Ballylreagent and compound 63 (see structure 323 in FIG. 11) were used toprepare the 328 a and 328 b (FIG. 11). The lack of a stereopredictivemodel for the formation of large rings via RCM is exemplified by ourresults with the metathetical ring closure of substrates 328 a,b. SeeFIG. 11. Whereas the diastereomeric substrate 312 c gave the Z-olefin318Z (vide supra, Scheme 4) as the major isomer, 328 b fortuitouslyproduced the E-benzolactone 329 a with an impressive selectivity of 10:1when subjected to similar reaction conditions. To confuse the issue evenmore, an initial single experiment with the corresponding phenolic MOMether 328 b furnished benzolactones 329 b and 330 b with an erodedselectivity of 3:1 upon exposure to catalyst i. FIG. 11. In principle,RCM is a reversible reaction but due to the shorter lifetime (thermalinstability) of “first generation” Ru-alkylidene catalysts (e.g. i) andless efficient reaction with 1,2-disubstituted olefins (reactionproducts), kinetic product ratios can be expected.

[0238] In light of the above, a detailed study of the RCM of 328 a,bwith Ru-alkylidene pre-catalysts i and ii was conducted. See FIG. 13.N-Heterocyclic carbene ligated ruthenium alkylidene catalysts have beenindependently reported by three groups, cf.: (a) Huang, J.; Stevens, E.D.; Nolan, S. P.; Petersen, J. L. J. Am. Chem. Soc. 1999, 121,2674-2678. (b) Scholl, M.; Trnka, T. M.; Morgan, J. P.; Grubbs, R. H.Tetrahedron Lett. 1999, 40, 2247-2250. (c) Ackermann, L.; Fürstner, A.;Weskamp, T.; Kohl, F. J.; Herrmann, W. A. Tetrahedron Lett. 1999, 40,4787-4790.

[0239] It is clear from these results that both catalysts are equallyefficient in performing the desired transformation, albeit withdifferent degrees of E/Z-selectivity. Also, both differentiallyprotected substrates 328 a,b gave identical E/Z-ratios under identicalreaction conditions. Whereas the pre-catalyst i provided a 9:1 (E:Z)ratio with only a slight erosion of selectivity over time, pre-catalystii produced a lower 67:33 ratio, which remains constant over time. Fromthese results, we conclude that RCM with catalyst i kinetically inducesthe formation of the desired E-isomers 329 a,b with relatively highselectivity. The ratio observed with catalyst ii is deemed to be athermodynamic one based on the following observations: (1) the ratiodoesn't change over time, (2) an identical product ratio is observed forthe formation of benzolactones 329-330 from both our precursors (328a,b), and (3) upon resubmission of either geometrically pure 329 a or330 a to the same reaction conditions, an identical 67:33 mixture of 329a: 330 a was formed.

[0240] Having secured a viable sequence to the benzolactone core ofsalicylihalamide A, we turned our attention to the installation of theacylated enamine side chain. Towards this end, the p-methoxybenzyl etherin 329 a (or 329 b) was oxidatively removed (DDQ) and the resultingalcohol 331 a was oxidized with Dess-Martin periodinane (FIG. 13).Engagement of the resulting aldehyde 332 in a Horner-Wadsworth-Emmons(HWE) homologation with trimethyl phosphonoacetate provided methyl ester334 a as an inseparable mixture of E/Z-isomers in a ratio of 4:1. Afterremoval of the ether protecting groups with BBr₃, the correspondingE-methyl ester was separated by flash chromatography, hydrolyzed(Ba(OH)₂.8H₂O) and extensively silylated with excess TBSCI to delivercarboxylic acid 337. The overall yield for the transformation of 332 to337 was a disappointing 20-30%. Therefore, a series of optimizationexperiments were performed that quickly led to the use of allyldiethylphosphonoacetate, delivering allyl ester 334 b with 18:1selectivity for the E-isomer. Subsequent deprotection, bis-silylationand Pd-catalyzed removal of the allyl ester improved the overall yieldof 337 dramatically (75% from aldehyde 332). Transformation intoacylazide 338 ((PhO)₂P(O)N₃, NEt₃, PhMe) preceded a Curtiusrearrangement induced by heat (PhH, 80° C., 6 h). Although thecorresponding vinylic isocyanate 339 was stable to chromatographicpurification, it was usually engaged in the next reaction withoutpurification. See FIG. 15.

[0241]FIG. 15 shows that final carbon-carbon bond formation proceededsmoothly via the addition of hexadienyllithium, prepared in situ frombromide 340 via metal-halogen exchange (t-BuLi, Et₂O), to a −78° C.solution of isocyanate 339. Compound 341 was obtained as an inseparablemixture of 22-Z and 22-E isomers together with a chromatographically(silicagel) more mobile fraction containing a mixture of E/Z-dimericcompounds 342. Careful fluoride-assisted desilylation was best performedwith a buffered solution of commercially available HF-pyridine inTHF/pyridine. At this point, geometrical isomers 301 a/ 301 c and343/344 could be separated by semi-preparative HPLC.

[0242] In contrast to the current study, we had initially synthesizedent-301 a based on the absolute configuration reported for the naturalproduct. Although this synthetic material was found to be identical tonatural salicylihalamide A according to NMR ([D₆]benzene and[D₄]methanol), IR, UV, and co-elution on HPLC (2 different solventsystems), and TLC (3 different solvent systems), the signs of theoptical rotations of synthetic ent-301 a ([α]²³ _(D)=+20.8; c=0.12;MeOH) and the natural product (reported: [α]²³ _(D=)−35; c=0.7; MeOH),were opposite. Moreover, synthetic ent-301 a was devoid of growthinhibitory activity when screened against the NCI 60-cell line panel. Atthis point we had the fortune that p-bromobenzoate derivative 348provided crystals suitable for X-ray diffraction studies, confirming theabsolute configuration of our synthetic lactones (FIG. 11(a) & (b)).Based on all the available evidence, the absolute configuration ofnatural (−)-salicylihalamide A was assigned to be as drawn in 301 a.Unequivocal proof for the absolute stereochemistry of naturalsalicylihalamide ultimately came from biological characterization ofsynthetic 301 a, which provided a differential cytotoxicity profileundistinguishable from the natural product in the NCI-60 cell linepanel. It is noted that careful examination of the ¹H-NMR spectra ofnatural salicylihalamide A indicates the presence of a minor contaminantwith an identical spectroscopic signature to our synthetic Z,E-isomer 1c (see FIG. 25 and FIG. 27). This contaminant could be a natural isomerof salicylihalamide A, or the result of isomerization during theisolation/purification procedure.

[0243] Structural variants with a suitable reporter were thenorchestrated towards side-chain modifications, emanating from a common,naturally configured isocyanate 339, now accessible in 20 steps (longestlinear sequence) and 21-25% overall yield. Thus, compound 346 and thecorresponding dimer 345 were prepared by addition of hexyllithium(instead of hexadienyllithium) to isocyanate 339 followed by finaldeprotection (FIG. 15). An inverse addition of isocyanate 339 to a coldsolution (−78° C.) of organolithium nucleophiles was next explored inorder to suppress dimer formation. Indeed, preparation of alkynoylenamine derivatives 349/350 followed this procedure, and no trace ofdimer formation was detected (FIG. 15). Alternatively, carbamates orthiocarbamates 351-354 are obtained in an operationally simplifiedprocedure by heating the acylazide 338 in the presence of theappropriate alcohol or thiol (FIG. 15).

[0244] A cell-based assay looking for growth inhibition of the SK-MEL 5human melanoma cell line was performed. Side chain modified analogs343-346 and 349-352 all retain cytostatic and cytotoxic properties atsimilar concentrations than the parent compound. See Table 3 below.TABLE 3 Growth inhibitory properties of selected compounds against thehuman melanoma cell line SK-MEL-5^(a) Compound GI₅₀ (μM) Compound GI₅₀(μM) Compound GI₅₀ (μM) 30a/1c 0.03 335b >20 343 0.04 344 0.1 345 0.6346 0.38 349 0.3 350 0.3 351 0.5 352 0.45 353 >20 354 1.5 355 >20 361 8362 >20 365 >20 366 1.0 367 >20 Apicularen A 0.006 235 0.06 236 0.9 2370.45 238 7.5 239 0.5

[0245] In addition, the ability of compounds 323 a/c and 343 to inhibitgrowth of a number of tumor cell lines was tested and compared toTAXOL's ability to inhibit growth of these tumor cell lines. The dataare indicated below in Table 4. TABLE 4 GI₅₀ (nM) GI₅₀ (nM) GI₅₀ (nM)Cell line 323a/c 343 TAXOL H1299 3.2 5.0 5.0 H2009 2.0 1.6 5.0 H358 1.610.0 5.0 H2058 3.2 6.3 5.0 H175 0.2 0.2 5.0 H1264 31.6 31.6 5.0

[0246] This indicates that the dienamide is not acting as a Michaelacceptor for biological relevant nucleophiles (346, 351-352) and thatsubstantial sterically demanding modifications can be accommodatedwithout abrogating biological activity (343-345). The Vacuolar ATPasewas identified as a putative target of salicylihalamide Alt hassubsequently been confirmed that the potent V-ATPase inhibitory activityof our synthetic salicylihalamides (see Table 5 below) and identifiedthe membrane spanning Vo proton channel as the binding site forsalicylihalamide A. The data of Table 5 was acquired using a purifiedsystem. Others in the art typically use crude membrane preparations toassay V-ATPase activity. Such a crude assay does not necessarily reflectV-ATPase specific activity. TABLE 5 Inhibition of purified reconstitutedV-ATPase from bovine brain^(a) Compound IC₅₀ (nM) Compound IC₅₀ (nM)Compound IC₅₀ (nM) 301a/301c 0.34 335b 230 343 0.37 344 1.2 345 3.0 3460.8 349 0.75 350 1.0 351 0.2 352 1.8 353 >1,800 354 >2,500 355 >1,000361 9.3 362 75.7 365 ND 366 300 367 180 Apicularen A <1.0 (+)− 270Bafilomycin A 3.1

[0247] In light of this, we prepared analogs 353/354 incorporating alipophilic cholesteryl or famesyl anchor hoping to target thesederivatives to the membrane (FIG. 15).

[0248] Side chain modified analogs that lack salicylihalamide'scharacteristic N-acyl enamine functionality are attractive candidatesfor the following reasons: (1) they are expected to confer increasedacid stability; (2) they can potentially be prepared via shortersequences; and (3) they would answer an important question related tothe functional role of the N-acyl enamine moiety. Octanoate 355, acompound with identical chain length and similar hydrophobicity thansalicylihalamide, is representative of this class of compounds and wasprepared from alcohol 331 via a Mitsunobu esterification followed bydeprotection (FIG. 17). However, both octanoate 355 and allyl ester 335b (FIG. 13) were completely inactive in the cell-based assay and 2-3orders of magnitude less potent than salicylihalamide in the in vitroV-ATPase assay.

[0249] At this point, we became entertained with the possibility thatsalicylihalamides could form a covalent complex with a putative bindingprotein through capture of an activated N-acyliminium ion by anucleophilic amino acid residue (FIG. 17), which could explain the lossof biological activity for derivatives 355/335 b.

[0250] A minimally perturbed probe to test this hypothesis wasenvisioned to arise from a simple saturation of the enamine double bondof biologically active salicylihalamide derivative 351. However, directhydrogenation of 351 also saturated the endocyclic double bond toproduce 362 (FIG. 18). Because there was no obvious short solution tothis chemoselectivity problem, a control reagent 361 was prepared as aprobe to investigate independently the effect of endocyclic double bondsaturation on biological activity (FIG. 18). Our point of departureentailed a hydrogenation of 329 a or 330 a with concomitant removal ofthe p-methoxybenzyl (PMB) ether. Subsequent conversion of 356 to 361took full advantage of the chemistry outlined for the preparation of 351without complication. Hydrogenation of this material also yielded thefully saturated salicylihalamide derivative 362. In vivo, N-carbamoylenamine derivative 361 retained a significant, although attenuated,level of growth inhibitory activity whereas the enamine to aminepermutation (361→362) completely abolished antiproliferative potential.In the in vitro V-ATPase assay, inhibition of proton pumping alsodecreases in the order 351 (IC₅₀ 0.2 nM)>361 (IC₅₀ 9.3 nM) >362 (IC₅₀75.7 nM). See Table 3 above. Because it was demonstrated thatsalicylihalamide is a reversible inhibitor of V-ATPase, it is likelythat other factors, perhaps increased conformational flexibility, areresponsible for the decreased biological activity of 362 (and to alesser extend for 361).

[0251] It became apparent from these studies that a photo-activatablesalicylihalamide reagent had to be developed in order to map thesalicylihalamide binding site on V-ATPase in molecular detail. Althoughthe potent in vivo and in vitro biological activity ofsalicylihalamide-based dimers 343-345 points to a potential site forattachment, the phenolic and secondary hydroxyls were also investigatedas a handle for derivatization (Scheme 10). Staring with bis-TBSderivative 336, selective deprotection of the phenolic (TBAF, THF, 0°C., 91%) or secondary TBS ether (aq. HCl, 91%) was followed bybenzoylation to furnish benzoates 363 (%) and 364 (50%) respectively.Together with compound 334 d, these materials were independentlyelaborated to bis- and mono-protected forms of salicylihalamidederivative 351, namely compounds 365-367. See FIG. 19. For the V-ATPaseactivity of 365-367 see Table 5 above

Example 8 Experimental Procedures for the Synthesis of SyntheticSalicylihalalmides and Characterization Data: I. General Techniques

[0252] Unless noted otherwise, commercially available materials wereused without further purification. All solvents used were of HPLC- orACS-grade. Solvents used for moisture sensitive operations weredistilled from drying agents under a nitrogen atmosphere: Et₂O and THFfrom sodium benzophenone ketyl; benzene and toluene from sodium; CH₂Cl₂,CH₃CN, NEt₃ and pyridine from CaH₂.

[0253] All moisture sensitive reactions were carried out under anitrogen atmosphere with magnetic stirring. Flash chromatography (FC)was performed using E Merck silicagel 60 (240-400 mesh) according to theprotocol of Still, Kahn, and Mitra (J. Org. Chem. 1978, 43, 2923). ThinLayer chromatography was performed using precoated plates purchased fromE. Merck (silicagel 60 PF254, 0.25 mm) that were visualized using aKMnO₄ or Ce(IV) stain.

[0254] Nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃,unless otherwise specified, on either a Varian Inova-400 or Mercury-300spectrometer at operating frequencies of 400/300 MHz (¹H NMR) or 100/60MHz (¹³C NMR). Chemical shifts (δ) are given in ppm relative to residualsolvent (usually chloroform; δ 7.27 for ¹H NMR or δ 77.25 for protondecoupled ¹³C NMR), and coupling constants (J) in Hz. Multiplicity istabulated as s for singlet, d for doublet, t for triplet, q forquadruplet, and m for multiplet, whereby the prefix app is applied incases where the true multiplicity is unresolved, and br when the signalin question is broadened.

[0255] Infrared spectra were recorded on a Perkin-Elmer 1000 series FTIRwith wavenumbers expressed in cm⁻¹ using samples prepared as thin filmsbetween salt plates. High-resolution mass spectra (HRMS) were recordedat the NIH regional mass spectrometry facility at the University ofWashington, St. Louis, Mo. Combustion analyses were performed by.Optical rotations were measured at 20° C. on a Perkin-Elmer 241 MCpolarimeter.

II. Experimental Procedure

[0256]

[0257] To a stirred solution of alcohol 304 (3.71 g, 8.57 mmol) in Et₂O(60 mL) was added p-methoxybenzyltrichloroacetimidate (4.3 g, 17.14mmol) followed by trifluoromethanesulfonic acid (2.3 μL, 0.026 mmol) andthe mixture was stirred for 2 h at rt. The mixture was quenched withaqueous sat. NaHCO₃ and extracted with Et₂O (3×). The combined organiclayers were washed (brine), dried (Na₂SO₄) and concentrated.Purification by FC yielded 3.4 g (72%) of p-methoxybenzyl ether 305 as acolorless oil. 305: [α]_(D)=−32.8 (CHCl₃, c 1.18); IR 2956, 2857, 1697,1613, 1514, 1331, 1250, 1136, 1096, 836, 777 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 7.26 (2H, d, J=8.4 Hz), 6.85 (2H, d, J=8.4 Hz), 4.55 (1H, d,J=10.8 Hz), 4.43 (1H, d, J=10.8 Hz), 4.17−4.11 (1H, m), 3.88 (1H, app.t,J=6.4 Hz), 3.79 (3H, s), 3.70 (2H, app.t, J=6.4 Hz), 3.50 (1H, d, J=13.6Hz), 3.43 (1H, d, J=13.6 Hz), 3.02 (1H, dd, J=6.4, 16.0 Hz), 2.98 (1H,dd, J=6.4, 16.0 Hz), 2.05-2.12 (2H, m), 1.75-1.96 (5H, m), 1.32-1.43(2H, m), 1.14 (3H, s), 0.96 (3H, s), 0.89 (9H, s), 0.04 (6H, s); ¹³C NMR(75 MHz, CDCl₃) δ 16.9, 159.2, 130.9, 129.5, 113.8, 72.9, 71.4, 65.3,59.6, 55.4, 53.1, 48.5, 47.9, 44.8, 41.2, 38.7, 38.0, 33.0, 26.6, 21.0,20.0, 18.4, −5.1, −5.2; MS (CI) 550 ([M-OMe]⁺, 2), 121 (100). HRMS Calcdfor C₂₉H₄₈NO₆SSi (MH⁺): 566.2972. Found: 566.2956.

[0258] To a −78° C. solution of p-methoxybenzyl ether 305 (2.4 g, 4.34mmol) in CH₂Cl₂ (40 mL) was added dropwise DIBAL-H (1.0 M in CH₂Cl₂, 5.2mL). After stirring for 1 h at −78° C., MeOH (2 mL) was added andstirring was continued for 5 min after which the cooling bath wasremoved and solid NaSO₄.10H₂O was added portionwise. After stirring for1 h at rt, the solid was removed by filtration and washed several timeswith EtOAc. The filtrate was concentrated and the residue partiallypurified to remove most of the bornanesultam auxiliary. The crudealdehyde 306 (˜1 g) was used in the next step without furtherpurification.

[0259] To a stirred solution of (2R)-N-(4′-pentynoyl)bomanesultam (950mg, 3.22 mmol) in CH₂Cl₂ (20 mL) at −78° C. was added TiCl₄ (343 μL,3.13 mL) and i-Pr₂NEt (0.56 mL, 3.22 mmol). After stirring for 1 h atthe same temperature, a solution of aldehyde 306 (˜1 g) in CH₂Cl₂ (2 mL)was added. After stirring for 2 h at −78° C., aqueous sat. NH₄Cl wasadded and an extraction was performed with EtOAc (3×). The combinedorganic layers were washed (brine), dried (Na₂SO₄) and concentrated. Theresidue was purified by FC (15% EtOAc/hexanes) yielding 1.33 g (74%) ofaldol product 307 as a white foam. 307: [α]_(D)=−54.7 (CHCl₃, c 1.39);IR 3480, 2956, 2857, 1694, 1613, 1514, 1334, 1250, 1095, 836, 777 cm⁻¹;¹H NMR (400 MHz, CDCl₃) δ 7.25 (2H, d, J=8.4 Hz), 6.84 (2H, d, J=8.4Hz), 4.49 (1H, d, J=10.8 Hz), 4.45 (1H, d, J=10.8 Hz), 4.26-4.32 (1H,m), 3.70-393 (2H, m), 3.78 (3H, s), 3.62-3.73 (2H, m), 3.52 (1H, d,J=2.4 Hz), 3.50 (1H, d, J=13.6 Hz), 3.42 (1H, d, J 13.6 Hz), 3.24 (1H,ddd, J=4.8, 7.2, 7.2 Hz), 2.84 (1H, ddd, J=2.8, 7.2, 17.2 Hz), 2.70 (1H,ddd, J=2.4, 4.8, 17.2 Hz), 2.10-2.17 (1H, m), 2.04 (1H, dd, J=7.6, 14.0Hz), 1.96 (1H, app.t, J=2.4 Hz), 1.84-1.94 (5H, m), 1.69-1.78 (1H, m),1.63 (1H, ddd, J=2.0, 6.4, 14.4 Hz), 1.30-1.42 (2H, m), 1.19 (3H, s),0.95 (3H, s), 0.88 (9H, s), 0.03 (6H, s); ¹³C NMR (75 MHz, CDCl₃) δ172.7, 159.3, 130.6, 129.6, 113.9, 80.5, 74.3, 71.2, 71.0, 67.2, 65.2,59.6, 55.3, 53.3, 49.5, 48.4, 47.9, 44.7, 38.5, 37.5, 36.8, 32.9, 26.6,26.1, 20.9, 20.0, 18.4, 18.3, −5.2; MS (CI) 648 [MH]⁺, 632, 590, 121(100). HRMS Calcd for C₃₄H₅₄NO₇SSi (MH⁺): 648.3390. Found: 648.3368.

[0260] To a solution of 307 (1.27 g, 1.96 mmol) in CH₂Cl₂ (10 mL) wasadded at −10° C. 2,6-lutidine (0.342 mL, 2.94 mmol), followed by TBSOTf(0.496 mL, 2.16 mmol). The solution was stirred for 2 h at −10° C. and 2h at rt followed by quenching with aqueous sat NH₄Cl. The aqueous layerwas extracted with EtOAc (3×) and the combined organics were washed(brine), dried (Na₂SO₄) and concentrated. The residue was purified by FC(10% EtOAc/hexanes) to give 1.33 g of silylether 308 as a colorless oil(90%). 308: [α]_(D)=−36.5 (CHCl₃, c 0.54); IR 2956, 2857, 1700, 1613,1515, 1332, 1250, 1100, 837, 777 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.33(2H, d, J=8.8 Hz), 6.85 (2H, d, J=8.8 Hz), 4.47 (2H, s), 4.37 (1H, d,J=5.6, 11.6 Hz), 3.88 (1H, dd, J=5.2, 7.6 Hz), 3.79 (3H, s), 3.65-3.75(3H, m), 3.49 (1H, d, J=13.6 Hz), 3.41 (1H, d, J=13.6 Hz), 3.37-3.42(1H, m), 2.80 (1H, ddd, J=2.4, 8.4, 16.8 Hz), 2.61 (1H, ddd, J=2.4, 3.6,16.8 Hz), 2.15-2.22 (1H, m), 2.07 (1H, dd, J=8.0, 14.0 Hz), 1.93 (1H, t,J=2.4 Hz), 1.77-1.92 (6H, m), 1.67 (1H, ddd, J=4.4, 6.4, 14.8 Hz),1.30-1.44 (2H, m), 1.20 (3H, s), 0.96 (3H, s), 0.91 (9H, s), 0.89 (9H,s), 0.07 (3H, s), 0.06 (9H, s); ¹³C NMR (75 MHz, CDCl₃) δ 171.2, 159.0,131.4, 129.4, 113.7, 81.6, 72.8, 70.6, 69.6, 68.8, 65.4, 59.9, 55.3,53.3, 50.2, 48.5, 47.9, 44.6, 42.2, 38.5, 37.7, 32.8, 26.6, 26.20,26.17, 20.8, 20.1, 18.5, 18.3, 18.0, −4.0, −4.1, −5.1; MS (CI) 762[MH]⁺, 713, 704, 514, 121 (100). HRMS Calcd for C₄₀H₆₈NO₇SSi₂ (MH⁺):762.4255. Found: 762.4246.

[0261] To a solution of silylether 308 (1.32 g, 1.73 mmol) in THF (60mL) was added LiEt₃BH (1.0 M solution in THF, 10.4 mL) at −78° C. Afterstirring for 2 h at −78° C. and 5 h at −40° C., aqueous NaOH (2.0N, 30mL) was added and the mixture was allowed to reach rt. Extraction(EtOAc, 3×), washing (brine), drying (Na₂SO₄) and concentration gave aresidue that was purified by FC (10% EtOAc/hexanes). Alcohol 309 wasobtained as a colorless oil (0.8 g, 84%). 309: [α]_(D)=−10.4 (CHCl₃, c1.01); IR 3500, 2955, 2930, 2858, 1614, 1515, 1251, 1094, 837, 776 cm⁻¹;¹H NMR (400 MHz, CDCl₃) δ 7.29 (2H, d, J=8.8 Hz), 6.87 (2 H, d, J=8.8Hz), 4.54 (1H, d, J=11.2 Hz), 4.41 (1H, d, J=11.2 Hz), 4.16-4.21 (1H,m), 3.81 (3H, s), 3.65-3.80 (5H, m), 2.52 (1H, app.t, J=5.6 Hz),2.13-2.19 (2H, m), 2.02-2.09 (1H, m), 1.99 (1H, app.t, J=2.4 Hz),1.69-1.90 (3H, m), 1.61 (1H, ddd, J=3.2, 7.6, 14.0 Hz), 0.91 (9H, s),0.90 (9H, s), 0.07 (6H, s), 0.066 (6H, s); ¹³C NMR (75 MHz, CDCl₃) δ159.3, 131.1, 129.3, 113.9, 83.1, 73.4, 71.3, 70.3, 69.8, 63.6, 59.7,55.5, 45.3, 38.8, 37.7, 26.18, 26.1, 18.5, 18.2, 17.1, −4.1, −5.1; MS(CI) 551 ([MH]⁺, 4), 493, 121 (100). HRMS Calcd for C₃₀H₅₅O₅Si₂ (MH⁺):551.3588. Found: 551.3576.

[0262] To a solution of alcohol 309 (0.78 g, 1.42 mmol) in CH₂Cl₂ (15mL) was added Et₃N (0.4 mL), DMAP (50 mg) and TsCl (0.407 g, 2.13 mmol).The mixture was stirred at rt for 16 h, diluted with Et₂O, washed (aq.NaHCO₃), dried (Na₂SO₄) and concentrated. The residue was purified by FC(5% EtOAc/hexanes) to yield 0.909 g (91%) of tosylate 310 as a colorlessoil. 310: [α]_(D)=−6.3 (CHCl₃, c 0.27); IR 2955, 2930, 2857, 1613, 1514,1363, 1250, 1178, 1098, 837, 777 cm⁻¹; ¹H NMR (400 MHz, CDCl₃), δ 7. 81(2H, d, J=8.4 Hz), 7.35 (2H, d, J=8.4 Hz), 7.29 (2H, d, J=8.8 Hz), 6.89(2H, d, J=8.8 Hz), 4.52 (1H, d, J=11.2 Hz), 4.39 (1H, d, J=11.2 Hz),4.06-4.19 (3H, m), 3.83 (3H, s), 3.63-3.77 (3H, m), 2.47 (3H, s), 2.30(1H, ddd, J=2.8, 7.2, 17.2 Hz), 2.21 (1H, ddd, J=2.8, 7.2, 17.2 Hz),2.09-2.16 (1H, m), 1.93 (1H, t, J=2.8 Hz), 1.78-1.87 (1H, m), 1.66-1.75(2H, m), 1.44 (1H, ddd, J=3.2, 7.6, 14.0 Hz), 0.93 (9H, s), 0.82 (9H,s), 0.09 (3H, s), 0.085 (3H, s), 0.04 (3H, s), 0.001 (3H, s); ¹³C NMR(75 MHz, CDCl₃) δ 159.2, 144.9, 133.0, 131.0, 130.0, 129.3, 128.2,113.9, 81.9, 73.3, 70.4, 70.2, 69.6, 68.6, 59.6, 55.5, 43.5, 39.1, 37.6,26.2, 26.0, 21.8, 18.5, 18.1, 16.3, −4.1, −4.4, −5.11, −5.13; MS (CI)705 [MH]⁺, 647, 568, 355, 121 (100). HRMS Calcd for C₃₇H₆₁NO₇SSi₂ (MH⁺):705.3677. Found: 705.3663.

[0263] To a solution of tosylate 310 (0.9 g, 1.28 mmol) in THF (13 mL)was added LiEt₃BH (1.0 M solution in THF, 6.4 mL) at −0° C. Afterstirring for 16 h at rt, aqueous NaOH (5%, 5 mL) was added. Extraction(Et2O, 3×), washing (brine), drying (Na₂SO₄) and concentration gave aresidue that was purified by FC (2% EtOAc/hexanes). Compound 311 wasobtained as a colorless oil (0.59 g, 86%). 311: [α]_(D)=−9.1 (CHCl₃, c1.06); IR 2955, 2930, 2858, 1614, 1515, 1472, 1250, 1095, 836, 775 cm⁻¹;¹H NMR (400 MHz, CDCl₃) δ 7.30 (2H, d, J=8.8 Hz), 6.88 (2H, d, J=8.8Hz), 4.53 (1H, d, J=11.2 Hz), 4.43 (1H, d, J=11.2 Hz), 3.98 (1H, app.dt,J=3.2, 8.0 Hz), 3.81 (3H, s), 3.66-3.78 (3H, m), 2.19 (1H, ddd, J=2.8,6.8, 16.8 Hz), 2.07 (1H, ddd, J=2.8, 8.4, 16.8 Hz), 1.96 (1H, t, J=2.8Hz), 1.86-1.93 (1H, m), 1.83 (1H, app.ddt, J=6.0, 6.0, 12.0 Hz), 1.74(1H, app.ddt, J=8.4, 8.4, 12.0 Hz), 1.64 (1H, ddd, J=4.0, 8.8, 14.0 Hz),1.50 (1H, ddd, J=4.0, 8.0, 14.0 Hz), 0.99 (3H, d, J=6.8 Hz), 0.92 (9H,s), 0.90 (9H, s), 0.08 (3H, s), 0.07 (6H, s), 0.06 (3H, s); ¹³C NMR (75MHz, CDCl₃) δ 159.2, 131.3, 129.3, 113.9, 83.9, 73.5, 72.1, 70.5, 69.2,59.8, 55.5, 55.4, 38.7, 38.5, 38.0, 26.2, 21.6, 18.5, 18.3, 14.9, −4.03,−4.04, −5.09, −5.1; MS (CI) 535 ([MH]⁺, 6), 534 (7), 533 (6) 519 (2),121 (100).

[0264] HRMS Calcd for C₃₀H₅₅O₄Si₂ (MH⁺): 535.3639. Found: 535.3548.

[0265]312 d: [α]_(D)=−10.7 (CHCl₃, c 0.3); IR 2960, 2926, 2860, 1517,1250, 1090, 833, 777 cm³¹ ¹; ¹H NMR (400 MHz, CDCl₃) δ 7.25 (2H, d,J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 5.69-5.82 (1H, in), 4.95-5.02 (2H,in), 4.50 (1H, d, J=10.8 Hz), 4.40 (1H, d, J=10.8 Hz), 3.87 (1H, ddd,J=3.2, 3.2, 8.8 Hz), 3.80 (3H, s), 3.60-3.79 (3H, m), 2.05 (1H, ddd,J=6.4, 6.4, 14.0 Hz), 1.64-1.89 (4H, m), 1.62 (1H, ddd, J=2.8, 8.8, 14.0Hz), 1.46 (1H, ddd, J=3.6, 8.8, 14.0 Hz), 0.90 (9H, s), 0.88 (9H, s),0.86 (3H, d, J=7.2 Hz), 0.06 (3H, s), 0.03 (3H, s), 0.026 (3H, s); ¹³CNMR (75 MHz, CDCl₃) δ 159.2, 138.0, 131.5, 129.2, 115.8, 113.9, 73.8,72.5, 70.6, 59.9, 55.5, 39.1, 38.1, 38.0, 37.4, 26.2, 18.5, 18.3, 14.2,−3.9, −4.1, −5.08, −5.10.

[0266] HRMS Calcd for C₃₀H₅₆O₄Si₂Li (MLi⁺): 543.3877. Found: 543.3875.

[0267]315: [α]_(D)=−23.7 (CHCl₃, c 0.6); IR 3528, 2956, 2930, 2858,1472, 1256, 1084, 836, 775 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 5.77 (1H,dddd, J=6.8, 6.8, 10.4, 16.8 Hz), 4.96-5.03 (2H, m), 3.96-4.04 (1H, m),3.75-3.91 (3H, m), 3.33 (1H, d, J=2.0 Hz), 2.12-2.20 (1H, m), 1.57-1.84(4H, m), 1.42-1.52 (2H, m), 0.90 (18H, s), 0.85 (3H, d, J=6.4 Hz), 0.10(3H, s), 0.07 (9H, s); ¹³C NMR (75 MHz, CDCl₃) δ 137.9, 115.8, 72.8,68.0, 62.4, 39.8, 39.2, 39.0, 37.9, 26.2, 26.1, 18.4, 18.3, 14.1, −4.2,−4.3, −5.26, −5.34. HRMS Calcd for C₂₂H₄₉O₃Si₂ (MH⁺): 417.3220. Found:417.3238.

[0268]314: HRMS Calcd for C₄₁H₆₈O₇Si₂Li (MLi⁺): 735.4664. Found:735.4635.

[0269]317 b: [α]_(D)=−8.5 (CHCl₃, c 0.33); IR 2957, 2930, 2857, 1728,1586, 1471, 1258, 1068, 836, 774 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.27(1H, dd, J=7.6, 8.0 Hz), 6.82 (1H, d, J=7.6 Hz), 6.77 (1H, d, J=8.0 Hz),5.92 (1H, dddd, J=6.8, 6.8, 9.6, 17.6 Hz), 5.78 (1H, dddd, J=7.2, 7.2,10.4, 17.7 Hz), 5.31-5.38 (1H, m), 4.96-5.09 (4H, m), 3.81 (3H, s),3.69-3.80 (3H, m), 3.33-3.37 (2H, m), 2.12-2.20 (1H, m), 1.74-2.00 (6H,m), 0.91 (3H, d, J=7.2 Hz), 0.902 (9H, s), 0.897 (9H, s), 0.06 (3H, s),0.06 (6H, s), 0.05 (3H, s), 0.04 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ167.7, 156.5, 138.4, 138.0, 136.5, 130.4, 124.4, 121.7, 116.7, 115.8,109.0, 72.6, 71.0, 59.9, 56.0, 38.1, 37.9, 37.6, 37.5, 36.4, 26.15,26.12, 18.5, 16.3, 15.5, −4.1, −4.2, −5.09, −5.13. HRMS Calcd forC₃₃H₅₈O₅Si₂Li (MLi⁺): 597.3983. Found: 597.3985.

[0270] To a solution of ester 317 b (13.5 g, 0.0203 mmol) in CH₂Cl₂ (1mL) was added at rt a solution of Cl₂[(Cy)₃P]₃Ru═CHPh (3.3 mg, 0.00406mmol) in CH₂Cl₂ (5mL) over a 3 h period via syringe pump. Afterevaporation of the solvent, the residue was purified by FC (2%EtOAc/hexanes) to give 2.2 mg of E-benzolactone 318-E (19%) and 9.4 mgof Z-benzolactone 318-Z (81%). 318-Z: [α]_(D)=−81.1 (CHCl₃, c 0.47); IR2928, 2856, 1718, 1588, 1471, 1283, 1255, 1082, 834, 772 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 7.29 (1H, t, J=8.0 Hz), 6.88 (1H, d, J=8.0 Hz), 7.76(1H, d, J=8.0 Hz), 5.37-5.46 (1H, m), 5.28-5.35 (2H, m), 3.92-3.97 (1H,m), 3.80 (3H, s), 3.59-3.79 (3H, m), 3.03 (1H, br d, J=14.4 Hz),2.20-2.38 (2H, m), 1.98-2.08 (1H, m), 1.74-1.88 (3H, m), 1.52-1.59 (1H,m), 0.91 (3H, d, J=7.2 Hz), 0.90 (9H, s), 0.87 (9H, s), 0.06 (3H, s),0.05 (3H, s), 0.04 (3H, s), 0.03 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ168.6, 156.2, 139.3, 130.5, 129.9, 128.4, 124.6, 121.9, 108.5, 77.4,72.0, 60.4, 55.8, 39.9, 36.7, 31.8, 29.9, 26.2, 26.1, 18.4, 18.3, −3.4,−4.0, −5.1. HRMS Calcd for C₃₁H₅₅O₅Si₂ (MH⁺): 563.3588. Found: 563.3572.318-E: [α]_(D)=−11.8 (CHCl₃, c 0.11); IR ¹H NMR (400 MHz, CDCl₃) 7.25(1H, dd, J=8.4, 8.0 Hz), 6.80 (1H, d, J=8.4 Hz), 6.78 (1H, d, J=8.0 Hz),5.42-5.54 (1H, m), 5.40 (1H, ddd, J=2.8, 8.0, 15.2 Hz), 5.23-5.28 (1H,m), 3.88 (1H, dd, J=6.0, 6.0 Hz), 3.80 (3H, s), 3.79-3.84 (1H, m),3.68-3.75 (2H, s), 3.14 (1H, br d, J=15.2 Hz), 2.39-2.46 (1H, m),2.20-2.30 (1H, m), 1.95-2.04 (1H, m), 1.76-1.90 (3H, m), 1.52-1.60 (1H,m), 0.93 (3H, d, J=7.2 Hz), 0.91 (9H, s), 0.88 (9H, s), 0.073 (3H, s),0.068 (3H, s), 0.04 (3H, s), 0.02 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ168.7, 156.8, 140.2, 130.5, 128.5, 124.5, 122.7, 109.4, 77.4, 71.8,60.3, 55.8, 38.5, 37.2, 36.3, 29.9, 26.2, 26.1, 18.5, 18.3, −3.8, −4.0,−5.0.

[0271] B-methoxy diisopinocampheylborane (13.7 g; 43.25 mmol) wasdissolved in Et₂O (80 mL), cooled to −78° C., followed by the dropwiseaddition of allylmagnesium bromide (1.0M in Et₂O, 41.7 mL). Afterstirring for 20 min at −78° C. and warming to rt over a 1.5 h period, aprecipitate formed (magnesium salts) and an additional 150 mL of Et₂Owas added. The precipitate was filtered under inert atmosphere andwashed with Et₂O (50 mL). The filtrate was cooled to −78° C. and asolution of aldehyde 319 (6.0 g, 30.89 mmol) in Et₂O (40 mL) was addeddropwise. After stirring for 4 h at the same temperature, the mixturewas treated with aqueous NaOH (2.0 N, 25 mL) and allowed to come to rt.Aqueous H₂O₂ (30%, 28 mL) was slowly added and stirring was continuedovernight. The aqueous layer was extracted with Et₂O (3×) and thecombined organics dried over MgSO₄ and concentrated in vacuo. FC(5%→10%→20% EtOAc/hexanes) afforded 6.0 g (25.4 mmol, 82%) of allylalcohol 320. 320: [α]_(D)=−3.9 (c 1.02, CHCl₃); IR 3445, 2936, 2863,1613, 1514, 1248, 1090, 1034 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ7.25 (2H, d,J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 5.83 (1H, dddd, J=7.2, 7.2, 10.2,17.0 Hz), 5.10 (1H, br d, J=17.0), 5.09 (1H, br d, J=10.2 Hz), 4.45 (2H,s), 3.82-3.89 (1H, m), 3.80 (3H, s), 3.68 (1H, app.dt, J=5.6, 9.6 Hz),3.61 (1H, ddd, J=5.6, 7.2, 9.6 Hz), 2.90 (1H, br s), 2.40 (2H, br app.t,J=7.0 Hz), 1.72-1.77 (2H, m); ¹³C NMR (75 MHz, CDCl₃) δ 159.3, 135.0,130.1, 129.4, 117.6, 114.0, 73.2, 70.7, 68.9, 55.5, 42.2, 36.1;

[0272] HRMS Calcd for C₁₄H₂₁O₃ (MH⁺): 237.1491. Found: 237.1495.

[0273] To a solution of allyl alcohol 320 (12 g, 50.78 mmol), imidazole(10.48 g, 152.34 mmol) and DMAP (200 mg) in DMF (200 mL) was addedtert.butyldimethylsilyl chloride (23.4 g, 152.34 mmol). After stirringfor 16 h at rt, the mixture was poured into water, extracted with Et₂O(3×), and the combined organics washed with brine, dried over MgSO₄ andconcentrated in vacuo. FC (2% EtOAc/hexanes) yields 16.7 g of silylether321 (94%) as an oil. 321: [α]_(D)=14.8 (c 1.51, CHCl₃); IR 2929, 2856,1612, 1514, 1248, 1093, 1040, 835, 774 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ7.25 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 5.75-5.86 (1H, m),5.00-5.05 (2H, m), 4.44 (1H, d, J=11.6 Hz), 4.38 (1H, d, J=11.6 Hz),3.86-3.91 (1H, m), 3.80 (3H, s), 3.50 (2H, app.t, J=6.4 Hz), 2.16-2.28(2H, m), 1.64-1.81 (2H, m), 0.88 (9H, s), 0.05 (3H, s), 0.04 (3H, s);¹³C NMR (75 MHz, CDCl₃) δ 159.2, 135.1, 130.8, 129.4, 117.1, 113.9,72.8, 69.2, 67.0, 55.5, 42.6, 37.0, 26.2, 18.4, −4.0, −4.3; MS (CI) 309([M-C₃H₅]⁺, 8), 121 (100). Anal. Calcd for C₂₀H₃₄O₃Si: C, 68.52; H,9.78. Found: C, 68.77; H, 9.78.

[0274] To a stirred solution of silylether 321 (22.2 g, 57.0 mmol) inacetone (220 mL) at 0° C. was added N-methyl-morpholine-N-oxide (50% wtin water, 16.0 g, 68 mmol) followed by OSO₄ (0.1 M in t-BuOH, 5.7 mL).The yellow solution was stirred for 4 h at 0° C. and 12 h at rt followedby concentration in vacuo. The residue was partitioned between EtOAc(200 mL) and brine (350 mL) to which aqueous HCl (1.0 N, 55 mL) andNa₂S₂O₃ (10.0 g) were added. Extraction of the aqueous layer with EtOAc(3×), drying of the combined organics over MgSO₄ and concentration invacuo, afforded an oil which was purified by FC (30%→50% EtOAc/hexanes)afforded 21 g of a mixture of diol diastereomers which was used in thenext step without further purification.

[0275] To a solution of the diol mixture (21 g, 54.6 mmol) in CH₂Cl₂(300 mL) was added at 0° C. silicagel-supported NaIO₄ (11.3 g, 0.582mmol/g silicagel). After stirring for 2 h at rt, the mixture wasfiltered, concentrated and purified by FC (10% EtOAc/hexanes) to give18.0 g of aldehyde 322 (53.04 mmol, 97%). 322: [α]_(D)=5.6 (c 2.38,CHCl₃); IR 2955, 2930, 2857, 1726, 1613, 1514, 1250, 1096, 1037, 837,777 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 9.79 (1H, app.t, J=2.4 Hz), 7.25(2H, d, J=9.2 Hz), 6.88 (2H, d, J=9.2 Hz), 4.43 (1H, d, J=11.2 Hz), 4.38(1H, d, J=11.2 Hz), 4.37 (1H, app.dt, J=6.0, 6.0 Hz), 3.81 (3H, s), 3.51(2H, app.t, J=6.0 Hz), 2.58 (1H, ddd, J=2.0, 5.6, 16.0 Hz), 2.51 (1H,ddd, J=2.8, 5.6, 16.0 Hz), 1.77-1.90 (2H, m), 0.86 (9H, s), 0.07 (3H,s), 0.06 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 202.1, 159.2, 130.4, 129.4,113.9, 72.9, 66.2, 65.9, 55.5, 51.3, 37.9, 26.0, 18.3, −4.27, −4.32;HRMS Calcd for C₁₉H₃₃O₄Si (MH⁺): 353.2148. Found: 353.2165.

[0276] To a solution of N-(4-pentenyl)-bomanesultam 323 (18.3 g, 61.27mmol) in CH₂Cl₂ (240 mL) was added at −78° C. TiCl₄ (6.44 mL, 58.71mmol), followed by the dropwise addition of ^(i)Pr₂NEt (11.2 mL, 63.82mmol). The deep red solution was stirred for 1 h at −78° C. and asolution of aldehyde 322 (18.0 g, 51.06 mmol) in CH₂Cl₂ (60 mL) wasadded dropwise. Stirring was continued for 2 h at the same temperatureand the mixture was quenched by the addition of phosphate buffer (pH7.0, 150 mL), saturated aqueous NaHCO₃ (150 mL) and H₂O. The aqueouslayer was extracted with CH₂Cl₂ (3×) and the combined organics weredried over MgSO₄ and concentrated in vacuo. The residue was purified byFC (15% EtOAc/hexanes) to give 30.4 g of aldol product 324 (46.8 mmol,92%). 324: [α]_(D)=45.9 (c 2.56, CHCl₃); IR 3515, 2956, 2856, 1689,1613, 1514, 1335, 1249, 1134, 1095, 1038, 836, 776 cm⁻¹; ¹H NMR (400MHz, CDCl₃) δ 7.25 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 5.79-5.90(1H, m), 5.08 (1H, br d, J=17.2 Hz), 4.97 (1H, br d, J=10.0 Hz), 4.42(1H, d, J=12 Hz), 4.38 (1H, d, J=12.0 Hz), 4.02-4.11 (2H, m), 3.85 (1H,app.t, J=6.4 Hz), 3.80 (3H, s), 3.51 (2H, app.t, J=6.4 Hz), 3.49 (1H, d,J=13.6 Hz), 3.40 (1H, d, J=13.6 Hz), 3.27 (1H, d, J=1.6 Hz), 3.21 (1H,app.dt, J=6.0, 8.0 Hz), 2.50-2.60 (2H, m), 1.99-2.05 (2H, m), 1.72-1.94(6H, m), 1.59 (1H, ddd, J=2.0, 6.4, 14.4 Hz), 1.30-1.39 (2H, m), 1.15(3H, s), 0.96 (3H, s), 0.87 (9H, s), 0.09 (3H, s), 0.07 (3H, s); ¹³C NMR(75 MHz, CDCl₃) δ 174.4, 159.1, 135.2, 130.8, 129.3, 117.5, 113.9, 72.8,69.5, 69.4, 66.7, 65.5, 55.5, 53.5, 50.6, 48.3, 47.9, 44.9, 41.8, 38.7,37.2, 33.6, 33.2, 26.7, 26.2, 21.2, 20.3, 18.3, −4.2. Anal. Calcd forC₃₄H₅₅NO₇SSi: C, 62.83; H, 8.53; N, 2.16. Found: C, 63.07; H, 8.67; N,2.89.

[0277] To a suspension of NaI (7.46 g, 49.23 mmol) in DME (45 mL) wasadded chloromethyl methyl ether (4.67 mL, 61.54 mmol). After stirringfor 15 min at rt a solution of aldol 324 (8.0 g, 12.31 mmol) in DME (65mL) was added followed by ^(i)Pr₂NEt (11.87 mL, 67.7 mL) and stirringwas continued for 12 h at reflux. The mixture was quenched by theaddition of saturated aqueous NaHCO₃ (60 mL) and water (250 mL). Theaqueous layer was extracted with Et₂O (4×), the combined organics washedwith HCl (1.0 M), and brine and dried over MgSO₄. After concentration invacuo, the residue was purified by FC (10% EtOAc/hexanes) to give 7.8 gof MOM-ether 325 (11.2, 91%). 325: [α]_(D)=75.3 (c 3.91, CHCl₃); IR2956, 2856, 1691, 1613, 1514, 1334, 1249, 1134, 1098, 1030, 837, 775cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.25 (2H, d, J=8.0 Hz), 6.85 (2 H, d,J=8.0 Hz), 5.77-5.88 (1H, m), 5.06 (1H, d, J=16.4 Hz), 4.96 (1H, d,J=10.0 Hz), 4.66 (1H, d, J=7.2 Hz), 4.61 (1H, d, J=7.2 Hz), 4.42 (1H, d,J=7.2 Hz), 4.38 (1H, d, J=7.2 Hz), 3.98 (1H, m), 3.85 (1H, dd, J=12.8,6.0 Hz), 3.78-3.83 (1H, m), 3.80 (3H, s), 3.54 (2H, app.t, J=6.4 Hz),3.47 (1H, d, J=14.0 Hz), 3.40 (1H, d, J=14.0 Hz), 3.28-3.34 (1H, m),3.32 (3H, s), 2.44-2.56 (2H, m), 1.80-2.06 (7H, m), 1.58-1.70 (2H, m),1.24-1.38 (2H, m), 1.14 (3H, s), 0.95 (3H, s), 0.87 (9H, s), 0.08 (3H,s), 0.04 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 172.9, 159.0, 135.1, 131.0,129.3, 117.5, 113.8, 97.3, 75.9, 72.6, 67.02, 66.98, 65.6, 56.5, 55.5,53.5, 50.2, 48.2, 47.9, 44.9, 42.9, 38.7, 36.3, 35.0, 33.2, 26.7, 26.2,21.2, 20.3, 18.4, −3.9, −4.5. HRMS Calcd for C₃₆H₅₉NO₈SSiLi (MLi⁺):700.3891. Found: 700.3879.

[0278] To a solution of MOM-ether 325 (5.2 g, 7.49 mmol) in THF (80 mL)at −78° C. was added dropwise LiEt₃BH (1.0 M in THF, 37.5 mL). Themixture was allowed to come to rt and stirring was continued for 3 h.The reaction was quenched by the addition of aqueous NaOH (2.0 N, 50 mL)and water (400 mL). The aqueous layer was extracted with Et₂O (3×),washed with HCl (1.0 N) and brine, and dried over MgSO₄. The resultingresidue was purified by FC (20% EtOAc/hexanes) to give 3.3 g of alcoholi (6.84 mmol, 91%). i: [α]_(D)=8.7 (c 1.0, CHCl₃); IR 3468, 2953, 2930,2857, 1613, 1514, 1250, 1094, 1037, 836, 776 cm⁻¹; ¹H NMR (400 MHz,CDCl₃), δ 7.24 (2H, d, J=8.4 Hz), 6.86 (2H, d, J=8.4 Hz), 5.71-5.81 (1H,m), 5.03 (1H, br d, J=16.8 Hz), 5.00 (1H, br d, J=9.5 Hz), 4.64 (1H, d,J=6.8 Hz), 4.60 (1H, d, J=6.8 Hz), 4.42 (1H, d, J=11.6 Hz), 4.38 (1H, d,J=11.6 Hz), 3.94-4.00 (1H, m), 3.80-3.85 (1H, m), 3.79 (3H, s),3.57-3.69 (2H, m), 3.52 (2H, app.t, J=6.4 Hz), 3.35 (3H, s), 2.75 (1H,br s), 1.91-2.06 (3H, m), 1.60-1.90 (4H, m), 0.88 (9H, s), 0.07 (3H, s),0.05 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 159.1, 136.8, 130.7, 129.4,116.5, 113.8, 96.4, 76.9, 72.9, 67.0, 66.7, 63.6, 56.3, 55.5, 42.9,38.9, 37.0, 31.9, 26.2, 18.3, −4.0, −4.3. Anal. Calcd for C₂₆H₄₆O₆Si: C,64.69; H, 9.60. Found: C, 64.66; H, 10.04.

[0279] A solution of alcohol i (2.0 g, 4.14 mmol), TsCl (3.99 g, 20.7mmol), DMAP (100 mg) and NEt₃ (3.46 mL, 24.86 mmol) in CH₂Cl₂ (40 mL)was stirred at rt for 16 h and at 35° C. for 5 h. The mixture was pouredinto water, extracted with Et₂O (3×) and the combined organics washedwith water and brine. After drying over MgSO₄ and concentration invacuo, the residue was purified by FC (10%→15% EtOAc/hexanes) to give2.4 g of tosylate ii (3.77, 91%). ii: [α]_(D)=17.3 (c 1.2, CHCl₃); IR2953, 2930, 2856, 1613, 1514, 1464, 1365, 1249, 1190, 1178, 1097, 1037,836, 776 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.79 (2H, d, J=8.4 Hz), 7.34(2H, d, J=8.4 Hz), 7.24 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz),5.60-5.70 (1H, m), 4.95-5.01 (2H, m), 4.50 (2H, s), 4.42 (1H, d, J=11.2Hz), 4.38 (1H, d, J=11.2 Hz), 4.10 (1H, dd, J=6.0, 9.6 Hz), 4.01 (1H,dd, J=5.6, 9.6 Hz), 3.88-3.94 (1H, m), 3.81 (3H, s), 3.67-3.71 (1H, m),3.50 (2H, app.t, J=6.0 Hz), 3.25 (3H, s), 2.45 (3H, s), 2.04-2.10 (2H,m), 1.98-2.04 (1H, m), 1.76-1.84 (1H, m), 1.59-1.68 (3H, m), 0.87 (9H,s), 0.03 (3H, s), 0.01 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 159.1, 144.7,135.7, 133.0, 130.6, 129.8, 129.3, 128.0, 117.1, 113.7, 96.3, 74.8,72.7, 69.9, 66.9, 66.6, 55.9, 55.4, 41.4, 39.1, 36.8, 31.2, 26.1, 21.8,18.2, −4.1, −4.4. Anal. Calcd for C₃₃H₅₂O₈SSi: C, 62.23; H, 8.23. Found:C, 60.34; H, 7.75.

[0280] To a stirred solution of tosylate ii (2.3 g, 3.61 mmol) in THF(70 mL) was added at 0° C. LiEt₃BH (1.0 M in THF, 14.4 mL). Afterstirring for 18 h at rt, aqueous NaOH (2.0 N, 12 mL) and water (400 mL)were added and an extraction was performed with Et₂O (3×). The combinedorganics were dried over MgSO₄, concentrated in vacuo and the residuepurified by FC (5% EtOAc/hexanes) to give 1.55 g of 326 (3.32 mmol, 92%)as an oil. 326: [α]_(D)=34.8 (c 2.45, CHCl₃); IR 2955, 2930, 2856, 1613,1514, 1249, 1096, 1038, 836, 775 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.25(2H, d, J=8.4 Hz), 6.86 (2H, d, J=8.4 Hz), 5.69-5.79 (1H, m), 4.99 (1H,br d, J=17.6 Hz), 4.98(1H, br d, J=9.6 Hz), 4.62 (1H, d, J=6.8 Hz), 4.57(1H, d, J=6.8 Hz), 4.43 (1H, d, J=11.2 Hz), 4.38 (1H, d, J 11.2 Hz),3.96-4.02 (1H, m), 3.80 (3H, s), 3.49-3.55 (3H, m), 3.33 (3H, s),2.01-2.10 (1H, m), 1.80-1.93 (3H, m), 1.61-1.70 (2H, m), 1.51 (1H, ddd,J=2.8, 8.4, 13.6 Hz), 0.88 (9H, s), 0.87 (3H, d, J=5.2 Hz), 0.06 (3H,s), 0.04 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 159.1, 137.4, 130.8, 129.3,116.0, 113.8, 95.9, 78.2, 72.8, 67.3, 66.9, 56.0, 55.4, 38.3, 37.5,36.8, 36.1, 26.2, 18.3, 14.4, −4.0, −4.4. Anal. Calcd for C₂₆H₄₆O₅Si: C,66.91; H, 9.93. Found: C, 66.52; H, 10.35.

[0281] To a solution of silylether 326 (1.49 g, 3.19 mmol) in THF (10mL) was added Bu₄NF (1.0 M in THF, 19.1 mL). After stirring for 12 h atrt, the mixture was quenched with water (150 mL). The aqueous layer wasextracted with Et₂O (3×) and the combined organics were dried over MgSO₄and concentrated in vacuo. The residue was purified by FC (25%EtOAc/hexanes) to give 1.1 g of alcohol 327 (3.12 mmol, 98%). 327:[α]_(D)=33.12 (c 2.6, CHCl₃); IR 3494, 2934, 1613, 1514, 1248, 1095,1036 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.26 (2H, d, J=8.4 Hz), 6.87 (2H,d, J=8.4 Hz), 5.69-5.80 (1H, m), 5.00 (1H, br J=15.6 Hz), 4.99 (1H, brd, J=10.8 Hz), 4.71 (1H, d, J=6.8 Hz), 4.63 (1H, d, J=6.8 Hz), 4.45 (2H,s), 3.91-3.98 (1H, m), 3.80 (3H, s), 3.56-3.70 (3H, m), 3.53 (1H, d,J=1.6 Hz), 3.39 (3H, s), 2.00-2.06 (1H, m), 1.81-1.96 (2H, m), 1.59-1.80(3H, m), 1.53 (1H, app.dt, J=3.6, 14.4 Hz), 0.89 (3H, d, J=6.8 Hz); ¹³CNMR (75 MHz, CDCl₃) δ 159.2, 137.1, 130.4, 129.4, 116.1, 113.9, 95.7,80.7, 73.0, 69.6, 68.0, 56.2, 55.5, 37.7, 37.2, 36.7, 35.7, 14.1. Anal.Calcd for C₂₀H₃₂O₅: C, 68.15; H, 9.15. Found: C, 68.30; H, 9.47.

[0282] To a stirred suspension of2,2-dimethyl-5-(trifluoromethanesulfonyl)-benzo[1,3]dioxin-4-one (iii)(2.0 g, 6.13 mmol), anhydrous LiCl (0.8 g, 18.4 mmol), Pd₂(dba)₃ (112mg, 0.12 mmol), and tri-(2-furyl)phosphine (115 mg, 0.49 mmol) in4-methyl-2-pyrrolidinone (10 mL) was added allyltributyltin (2.35 mL,7.36 mmol). After stirring for 48 h at rt, saturated aqueous KF (20 mL)was added and an extraction was performed with Et₂O (3×). The combinedorganics were dried over MgSO₄, concentrated in vacuo and the residuewas purified by FC (4% EtOAc/hexanes) to give 1.3 g of5-allyl-2,2-dimethyl-benzo[1,3]dioxin-4-one (iv) which was slightlyimpure.

[0283] To a stirred solution of allyl alcohol (2.25 mL, 32.8 mL) in THF(10 mL) was added dropwise EtMgBr (1.0M in THF, 29.8 mmol) at 0° C.After stirring for 15 min at rt, a solution of5-allyl-2,2-dimethyl-benzo[1,3]dioxin-4-one (iv) (1.3 g, 5.96 mmol) inTHF (5 mL) was added and stirring was continued for 5 h at rt and 2.5 hat 60° C. Diethyl ether (50 mL) was added and the mixture was pouredinto saturated aqueous NH₄Cl (20 mL) and water (50 mL). An extractionwas performed with Et₂O (3×) and the combined organics were dried overMgSO₄ and concentrated in vacuo. The residue was purified by FC (2%Et₂O/pentane) to give 1.2 g of allyl 6-allyl-2-hydroxybenzoate (v) (90%)as a colorless oil. v: IR 2918, 2850, 1663, 1607, 1450, 1248, 1219, 909,733 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.33 (1H, app.t, J=7.3 Hz), 6.88(1H, br d, J=7.3 Hz), 6.75 (1H, d, J=7.3 Hz), 5.92-6.09 (2H, m), 5.43(1H, br d, J=14.0 Hz), 5.33 (1H, br d, J=8.3 Hz), 5.02 (1H, br d, J=10.0Hz), 4.96 (1H, br d, J=16.5 Hz), 4.86 (2H, br d, J=6.7 Hz), 3.70 (2H, d,J=5.9 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 162.8, 143.0, 137.7, 134.6,131.5, 122.6, 119.8, 116.3, 115.6, 112.2, 66.7, 40.6; MS (EI) 218 (M⁺,10), 177 (24), 159 (100).

[0284] To a solution of allyl 6-allyl-2-hydroxybenzoate (v) (1.2 g, 5.5mmol) in acetone (12 mL) was added K₂CO₃ (0.84 g, 6.05 mmol) and Mel(1.04 mL, 16.5 mmol) and the mixture was stirred for 40 h at rt. Afterfiltration of the mixture through a short plug of silicagel, thefiltrate was concentrated in vacuo and the residue purified by FC (3%Et₂O/pentane) to give 1.25 of allyl 6-allyl-2-methoxybenzoate (via)(98%) as a colorless oil. via: IR 2918, 2850, 1733, 1715, 1585, 1363,1244, 1223, 914, 733 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.29 (1H, app.t,J=8.0 Hz), 6.83 (1H, d, J=7.6 Hz), 6.79 (1H, d, J=8.4 Hz), 5.97-6.06(1H, m), 5.86-5.96 (1H, m), 5.42 (1H, br d, J=17.2 Hz), 5.28 (1H, br d,J=10.4 Hz), 5.05 (1H, br d, J=16.8 Hz), 5.04 (1H, br d, J=10.0 Hz), 4.82(2H, br d, J 6.0 Hz), 3.82 (3H, s), 3.36 (2H, d, J=6.8 Hz); ¹³C NMR (75MHz, CDCl₃) δ 167.8, 156.6, 138.7, 136.4, 132.1, 130.6, 123.6, 121.8,118.8, 116.5, 109.2, 66.0, 56.1, 37.9; MS (EI) 232 (M⁺, 18), 191 (28),175(34), (147 (50), 132 (100), 115 (58). Anal. Calcd for C₁₄H₁₆O₃: C,72.39; H, 6.94. Found: C, 72.10; H, 6.87. vib (MOM): IR 2956, 2928,2853, 1732, 1636, 1589, 1466, 1256, 1153, 1031, 919 cm⁻¹; ¹H NMR (400MHz, CDCl₃) δ 7.26 (1H, dd, J=7.6, 8.4 Hz), 7.01 (1H, d, J=7.6 Hz), 6.88(1H, d, J=8.4 Hz), 6.01 (1H, ddt, J=5.6, 10.0, 17.2 Hz), 5.90 (1H, ddt,J=6.8, 10.4, 16.8 Hz), 5.42 (1H, br d, J=17.2 Hz), 5.28 (1H, br d,J=10.0 Hz), 5.17 (2H, s), 5.06 (1H, br d, J=16.8 Hz), 5.05 (1H, br d,J=10.4 Hz), 4.81 (2H, d, J=5.6 Hz), 3.46 (3H, s), 3.37 (2H, d, J=6.8Hz); ¹³C NMR (75 MHz, CDCl₃) δ 167.8, 154.2, 138.8, 136.5, 132.2, 130.7,124.7, 123.0, 118.9, 116.6, 112.9, 94.9, 66.0, 56.4, 37.8. HRMS Calcdfor C₁₅H18O₄Li (MLi⁺): 269.1365. Found: 269.1371.

[0285] To a stirred solution of allyl 6-allyl-2-methoxybenzoate (via)(1.01 g, 4.35 mmol) and Pd(Ph₃)₄ in THF (10 mL) was added morpholine(3.5 mL, 43.5 mmol). After stirring for 1 h at rt, the mixture wasconcentrated in vacuo and the residue purified by FC (2% MeOH/CH₂Cl₂containing 0.3% ACOH) to give 0.8 g of benzoic acid derivative 316 a(96%). 316 a: IR 2919, 2850, 1713, 1586, 1470, 1264, 910, 733 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 7.34 (1H, app.t, J=8.0 Hz), 6.89 (1H, d, J=7.6Hz), 6.85 (1H, d, J=8.4 Hz), 5.92-6.02 (1H, m), 5.06-5.12 (2H, m), 3.90(3H, s), 3.55 (2H, d, J=6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 172.1,156.9, 140.1, 136.6, 131.3, 122.5, 122.0, 116.5, 109.4, 56.4, 38.3; MS(EI) 192 (M⁺, 52), 177 (100). HRMS Calcd for C₁₁H₁₃O₃ (MH⁺): 193.0865.Found: 193.0864. 316 b: HRMS Calcd for C ₁₂H15O₄ (MH⁺): 223.0970. Found:223.0972.

[0286] To a stirred solution of alcohol 327 (795 mg, 2.255 mmol), acid316 a (650 mg, 3.38 mmol) and PPh₃ (azeotropically dried with benzene,956 mg, 3.61 mmol) in Et₂O (35 mL) was added diethylazodicarboxylate(DEAD, 0.57 mL, 3.61 mmol). After stirring for 12 h at rt and 4 h atreflux, the mixture was partitioned between water (200 mL) and Et₂O (100mL). The aqueous layer was extracted with Et₂O (2×) and the combinedorganics dried over MgSO₄ and concentrated in vacuo. The residue waspurified by FC (10% EtOAc/hexanes) to afford 1.04 g of ester 328 a (1.97mmol, 88%). 328 a: [α] _(D)=−2.44 (c 1.80, CHCl₃); IR 2932, 1725, 1585,1514, 1470, 1266, 1247, 1099, 1071, 1037, 915 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 7.28 (1H, app.t, J=7.6 Hz), 7.27 (2H, d, J=8.4 Hz), 6.86 (2H,d, J=8.4 Hz), 6.82 (1H, d, J=7.6 Hz), 6.77 (1H, d, J=8.8 Hz), 5.87-5.97(1H, m), 5.69-5.79 (1H, m), 5.40-5.47 (1H, m), 5.05 (1H, br d, J=9.0Hz), 5.03 (1H, br d, J=17.0 Hz), 4.96 (1H, br d, J=18.0 Hz), 4.91 (1H,br d, J=10.4 Hz), 4.73 (1H, d, J=6.8 Hz), 4.71 (1H, d, J=6.8 Hz), 4.47(1H, d, J=11.6 Hz), 4.42 (1H, d, J=11.6 Hz), 3.80 (3H, s), 3.79 (3H, s),3.70 (1H, app.quint, J=4.0 Hz), 3.55-3.65 (2H, m), 3.40 (3H, s), 3.35(2H, d, J=6.8 Hz), 1.97-2.11 (3H, m), 1.87-1.97 (1H, m), 1.77-1.87 (1H,m), 1.66-1.78 (2H, m), 0.90 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ167.9, 159.2, 156.3, 138.2, 137.2, 136.5, 130.6, 130.3, 129.3, 124.2,121.7, 116.5, 116.0, 113.9, 108.8, 97.2, 78.5, 72.9, 70.9, 66.7, 56.0,55.8, 55.5, 37.9, 37.5, 36.8, 35.6, 35.5, 14.0. Anal. Calcd forC₃₁H₄₂O₇: C, 70.70; H, 8.04. Found: C, 70.62; H, 8.32. ent-328 b (MOM):[α]_(D)=−3.2 (c 1.50, CHCl₃); IR 2932, 1726, 1585, 1513, 1465, 1249,1095, 1035 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.23-7.29 (3H, m), 7.03 (1H,d, J=8.0 Hz), 6.85-6.89 (3H, m), 5.93 (1H, dddd, J=6.0, 6.0, 10.4, 16.8Hz), 5.72 (1H, dddd, J=7.2, 7.2, 10.0, 16.8 Hz), 5.40-5.47 (1H, m), 5.18(1H, d, J=6.8 Hz), 5.13 (1H, d, J=6.8 Hz), 5.02-5.09 (2H, m), 4.95 (1H,br d, J=16.8 Hz), 4.92 (1H, br d, J=10.4 Hz), 4.73 (1H, d, J=6.8 Hz),4.70 (1H, d, J=6.8 Hz), 4.47 (1H, d, J=11.6 Hz), 4.42 (1H, d, J=11.6Hz), 3.80 (3H, s), 3.55-3.70 (3H, m), 3.44 (3H, s), 3.40 (3H, s), 3.36(2H, d, J=6.0 Hz), 1.70-2.12 (7H, m), 0.89 (3H, d, J=6.8 Hz); ¹³C NMR(75 MHz, CDCl₃) δ 167.9, 159.3, 154.0, 138.3, 137.3, 136.5, 130.7,130.3, 129.4, 125.1, 122.8, 116.7, 116.1, 113.9, 112.4, 97.0, 94.6,78.4, 72.9, 71.0, 66.6, 56.2, 55.9, 55.4, 37.8, 37.4, 36.7, 35.5, 35.3,13.9. HRMS Calcd for C₃₂H₄₄O₈Li (MLi⁺): 563.3196. Found: 563.3202.

[0287] To a flask charged with degassed CH₂Cl₂ (180 mL) was addedsimultaneously a solution of Cl₂[(Cy)₃P]₃Ru═CHPh (141 mg, 0.17 mmol) inCH₂CI₂ (30 mL) and a solution of ester 328 a (0.6 g, 1.14 mmol) inCH₂Cl₂ (50 mL) over a 4 h period via addition funnels. After theaddition was complete, the solvent was evaporated and the residuepurified by FC (15% EtOAc/hexanes) to give 465 mg of benzolactone 329 a(0.93 mmol, 81%) and 46 mg of the corresponding Z-isomer 330 a (0.092mmol, 8%). 329 a: [α] _(D)=−49.5 (c 1.30, CHCl₃); IR 2955, 2931, 2840,1724, 1613, 1585, 1514, 1468, 1275, 1249, 1098, 1071, 1040 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 7.27 (2H, d, J=8.4 Hz), 7.22 (1H, app.t, J=8.0 Hz),6.86 (2H, d, J=8.4 Hz), 6.78 (1H, d, J=8.0 Hz), 6.76 (1H, d, J=8.0 Hz),5.44-5.53 (2H, m), 5.34 (1H, br dd, J=9.2, 15.2 Hz), 4.88 (1H, d, J=6.8Hz), 4.79 (1H, d, J=6.8 Hz), 4.47 (2H, s), 4.15 (1H, dd, J=3.6, 9.2 Hz),3.79 (3H, s), 3.72 (1H, dd, J=9.6, 16.4 Hz), 3.71 (3H, s), 3.65 (2H,app.t, J=6.8 Hz), 3.43 (3H, s), 3.32 (1H, br d, J=16.4 Hz), 2.30 (1H, brd, J=14.0 Hz), 2.07-2.18 (1H, m), 2.02 (1H, app. ddt, J=6.8, 9.2, 14.4Hz), 1.91 (1H, dddd, J=4.0, 7.2, 7.2, 14.4 Hz), 1.76 (1H, dd, J=8.8,15.2 Hz), 1.71 (1H, app.td, J=11.6, 14.0 Hz), 1.45 (1H, dd, J=9.2, 15.2Hz), 0.86 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.3, 159.1,156.5, 139.2, 131.5, 130.8, 130.1, 129.2, 128.6, 124.7, 123.0, 113.9,109.3, 97.0, 79.4, 72.8, 72.5, 66.9, 55.8, 55.6, 55.5, 38.04, 38.0,36.7, 36.0, 34.3, 13.7; HRMS Calcd for C₂₉H₃₉O₇ (MH⁺): 499.2696. Found:499.2697. 330 a: [α] _(D)=−25.0 (c 0.6, CHCl₃); IR 2932, 1729, 1613,1598, 1514, 1469, 1250, 1115, 1065, 1038 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ7.29 (1H, app.t, J=7.6 Hz), 7.27 (2H, d, J=8.8 Hz), 6.85 (2H, d, J=8.8Hz), 6.82 (1H, d, J=6.4 Hz), 6.82 (1H, d, J=8.4 Hz), 5.53 (1H, app.dt,J=3.6, 9.6 Hz), 5.30-5.37 (2H, m), 4.78 (1H, d, J=6.8 Hz), 4.72 (1H, d,J=6.8 Hz), 4.46 (1H, d, J=11.2 Hz), 4.38 (1H, d, J=11.2 Hz), 3.97 (1H,dd, J=8.0, 15.2 Hz), 3.74-3.84 (1H, m), 3.80 (3H, s), 3.76 (3H, s),3.50-3.58 (2H, m), 3.39 (3H, s), 3.03 (1H, br d, J=14.0 Hz), 1.85-2.18(6H, m), 1.54-1.60 (1H, m), 0.93 (3H, d, J=6.4 Hz); ¹³C NMR (75 MHz,CDCl₃) δ 166.9, 159.1, 157.1, 140.1, 130.8, 130.7, 129.4, 129.3, 128.8,123.7, 122.8, 113.8, 109.5, 97.4, 77.9, 73.0, 72.0, 66.8, 56.1, 55.8,55.5, 36.7, 36.6, 35.8, 32.8, 32.2, 13.8. ent-329 b/ent-330 b (MOM): ¹HNMR (400 MHz, CDCl₃), major isomer only, δ 7.26 (2H, d, J=8.4 Hz), 7.20(1H, dd, J=8.4, 7.6 Hz), 7.03 (1H, d, J=8.4 Hz), 6.87 (2H, d, J=8.4 Hz),6.80 (1H, d, J=7.6 Hz), 5.44-5.56 (2H, m), 5.34 (1H, br dd, J=9.2, 15.6Hz), 5.06 (2H, s), 4.88 (1H, d, J=6.8 Hz), 4.79 (1H, d, J=6.8 Hz), 4.46(2H, s), 4.13 (1H, dd, J=3.2, 9.2 Hz), 3.72 (1H, dd, J=8.8, 16.0 Hz),3.66 (2H, app.t, J=6.8 Hz), 3.43 (3H, s), 3.39 (3H, s), 3.32 (1H, br d,J=16.0 Hz), 2.30 (1H, br d, J=14.0 Hz), 2.07-2.19 (1H, m), 1.86-2.06(2H, m), 1.76 (1H, dd, J=8.8, 15.2 Hz), 1.70 (1H, ddd, J=11.6, 11.6,14.0 Hz), 1.47 (1H, dd, J=9.6, 15.2 Hz), 0.87 (3H, d, J=7.2 Hz [minorZ-isomer at 0.93]). HRMS Calcd for C₃₀H₄₀O₈Li (MLi⁺): 535.2883. Found:535.2876.

[0288] To a solution of benzolactone 329 a (0.85 g, 1.70 mmol) in CH₂Cl₂(35 mL) and water (1.7 mL) was added DDQ (472 mg, 2.04 mmol). Afterstirring for 1.5 h at rt, the yellow slurry was poured into saturatedaqueous NaHCO₃ (10 mL) and water (100 mL) and an extraction wasperformed with EtOAc (4×). The combined organic layers were dried overMgSO₄ and concentrated in vacuo. The residue was purified by FC (50%EtOAc/hexanes) to give 617 mg of alcohol 331 (1.63 mmol, 96%). 331:[α]_(D)=−50.4 (c 1.21, CHCl₃); IR 3440, 2955, 1723, 1584, 1469, 1275,1039 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.24 (1H, app.t, J=8.0 Hz), 6.81(1H, d, J=8.4 Hz), 6.78 (1H, d, J=7.6 Hz), 5.45-5.54 (2H, m), 5.35 (1H,dddd, J=2.0, 2.0, 9.6, 15.6 Hz), 4.89 (1H, d, J=6.8 Hz), 4.82 (1H, d,J=6.8 Hz), 4.17 (1H, dd, J=3.6, 8.8 Hz), 3.82 (3H, s), 3.69-3.87 (3H,m), 3.46 (3H, s), 3.33 (1H, dddd, J=2.0, 2.0, 4.4, 16.4 Hz), 2.43 (1H,br s), 2.31 (1H, br d, J=14.0 Hz), 2.09-2.19 (1H, m), 1.80-1.98 (2H, m),1.79 (1H, dd, J=8.0, 15.6 Hz), 1.72 (1H, app.td, J=11.6, 14.0 Hz), 1.45(1H, dd, J=9.2, 15.6 Hz), 0.87 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz,CDCl₃) δ 168.3, 156.2, 139.3, 131.6, 130.2, 128.4, 124.4, 123.3, 109.4,97.2, 79.7, 73.2, 59.6, 55.9, 55.8, 39.2, 38.0, 36.2, 34.3, 13.7; HRMSCalcd for C₂₁H₃₁O₆ (MH⁺): 379.2121. Found: 379.2120. ent- (MOM): HRMSCalcd for C₂₂H₃₂O₇Li (MLi⁺): 415.2308. Found: 415.2323.

[0289] To a solution of alcohol 331 (337 mg, 0.89 mmol) in CH₂Cl₂ (30mL) was added Dess-Martin periodinane (0.94 g, 2.23 mmol). Afterstirring at rt for 3 h, saturated aqueous NaHCO₃ (5 mL) and water (100mL) was added and an extraction was performed with Et₂O. The combinedorganic layers were dried (MgSO₄) and concentrated after which theresidue was purified by FC (30% EtOAc/hexanes) to give 318 mg ofaldehyde 332 (0.845 mmol, 95%) as an oil. 332: [α]_(D)=−45.4 (c 1.55,CHCl₃); IR 2962, 2935, 1728, 1585, 1470, 1274, 1117, 1086, 1037 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 9.79 (1H, dd, J=1.6, 3.6 Hz), 7.23 (1H, app.t,J=8.0 Hz), 6.77 (1H, d, J=8.0 Hz), 6.75 (1H, d, J=8.0 Hz), 5.09 (1H,ddd, J=3.6, 8.8, 10.0 Hz), 5.49 (1H, br dd, J=11.6, 15.2 Hz), 5.34 (1H,br dd, J=10.0, 15.2 Hz), 4.92 (1H, d, J=7.2 Hz), 4.85 (1H, d, J=7.2 Hz),4.20 (1H, dd, J=3.6, 9.2 Hz), 3.74 (3H, s), 3.71 (1H, dd, J=10.0, 16.4Hz), 3.47 (3H, s), 3.31 (1H, br d, J=16.4 Hz), 2.77 (1H, ddd, J=3.6,10.0, 16.4 Hz), 2.53 (1H, ddd, J=1.6, 3.6, 16.4 Hz), 2.31 (1H, br d,J=14.0 Hz), 2.10-2.21 (1H, m), 1.83 (1H, dd, J=8.8, 15.2 Hz), 1.71 (1H,app.td, J=11.6, 14.0 Hz), 1.45 (1H, dd, J=9.2, 15.2 Hz), 0.87 (3H, d,J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 200.5, 168.0, 156.6, 139.0, 131.5,130.3, 128.7, 123.9, 122.7, 109.2, 97.2, 79.4, 69.8, 55.9, 55.5, 49.4,37.5, 37.87, 35.9, 34.5, 13.6.

[0290] A mixture of 329 a (120 mg, 0.317 mmol) and 10% Pd/C (30 mg) inMeOH (5 mL) was stirred under an atmosphere of H₂ for 3 h at rt.Filtration, concentration and purification by FC (50% EtOAc in hexanes)delivered 99 mg of alcohol 356 (82%). Alternatively, a mixture of alkene330 a (111 mg, 0.223 mmol), ammonium formate (100 mg) and 10% Pd/C (100mg) in MeOH (6 mL) was refluxed for 6 h. Filtration, concentration andpurification of the residue by FC (50% EtOAc/hexanes) delivered 57 mg ofalcohol 356 (0.15 mmol, 67%). 356: [α]_(D)=−35.6 (c 1.0, CHCl₃); IR3451, 2933, 1724, 1582, 1470, 1265 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.26(1H, t, J=8.0 Hz), 6.79 (1H, d, J=7.6 Hz), 6.79 (1H, d, J=8.4 Hz), 5.52(1H, app.q, J=8.0 Hz), 4.76 (2H, s), 5.83 (3H, s), 3.70-84 (3H, m), 3.44(3H, s), 2.80-2.91 (1H, m), 2.42-2.51 (2H, m), 1.98-2.06 (1H, m),1.87-1.95 (3H, m), 1.60-1.78 (3H, m), 1.42-1.56 (2H, m), 1.18-1.30 (2H,m), 0.91 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.7, 156.5,141.5, 130.8, 123.6, 122.4, 109.0, 96.7, 71.6, 58.9, 56.07, 55.97, 39.0,35.8, 34.1, 31.8, 26.1, 15.4; HRMS Calcd for C₂₁H₃₃O₆ (MH⁺): 381.2277.Found: 381.2274.

[0291] According to the procedure described for the synthesis of 332,alcohol 356 (52 mg, 0.137 mmol) was oxidized with Dess-Martinperiodinane (231 mg, 0.547 mmol), delivering 42 mg of aldehyde 357(81%).

[0292] To a suspension of NaH (60% in mineral oil, 74 mg, 1.84 mmol) inTHF (15 mL) was added allyl diethylphosphonoacetate (0.436 mL, 2.0 mmol)at 0° C. After stirring for 20 min at the same temperature, a solutionof aldehyde 332 (302 mg, 0.802 mmol) in THF (5 mL) was added andstirring was continued for 1 h at 0° C. Saturated aqueous NaHCO₃ (5 mL)and water (100 mL) was added and an extraction was performed with Et₂O(3×). After concentration of the organic layers, drying (MgSO₄) andconcentration in vacuo, the residue was purified by FC (20%EtOAc/hexanes) to yield 343 mg of allyl ester 334 b (0.75 mmol, 93%).Esters 334 a-c were prepared using the same procedure withtrimethylsilyl dimethylphosphonoacetate, t-butyldimethylphosphonoacetate and trimethyl phosphonoacetate respectively.334 b: [α] _(D)=−95.0 (c 2.49, CHCl₃); IR ¹H NMR (400 MHz, CDCl₃) δ 7.23(1H, app.t, J=8.0 Hz), 7.14 (1H, ddd, J=6.0, 8.0, 15.6 Hz), 6.81 (1H, d,J=8.4 Hz), 6.75 (1H, d, J=7.2 Hz), 5.95 (1H, d, J=15.6 Hz), 5.90-6.00(1H, m), 5.32-5.52 (3H, m), 5.3 (1H, td, J=1.2, 17.0 Hz), 5.24 (1H, td,J=1.2, 10.4 Hz), 4.89 (1H, d, J=6.8 Hz), 4.81 (1H, d, J=6.8 Hz), 4.64(2H, dd, J=1.2, 5.6 Hz), 4.16 (1H, dd, J=3.6, 9.6 Hz), 3.85 (3H, s),3.71 (1H, dd, J=9.6, 16.4 Hz), 3.45 (3H, s), 3.32 (1H, br d, J=16.4 Hz),2.65 (1H, ddd, J=6.0, 7.6, 15.6 Hz), 2.47 (1H, ddd, J=4.0, 8.0, 15.6Hz), 2.27-2.36 (1H, m), 2.08-2.19 (1H, m), 1.76 (1H, dd, J=8.8, 15.6),1.70 (1H, app.dt, J=11.6, 14.0 Hz), 1.44 (1H, dd, J=9.6, 15.6 Hz), 0.86(3H, d, J=6.4 Hz); HRMS (FAB) Calcd for C₂₆H₃₅O₇ (MH⁺): 459.2383. Found:459.2386. ent-334 c (t-butyl): [α]_(D)=+68.6 (c 0.35, CHCl₃); IR 2933,1713, 1653, 1587, 1473, 1277, 1250, 1153, 1117, 1040 cm⁻¹; ¹H NMR (400MHz, CDCl₃) δ 7.23 (1H, dd, J=7.6, 8.4 Hz), 6.99 (1H, ddd, J=6.0, 8.4,15.6 Hz), 6.81 (1H, d, J=8.4 Hz), 6.75 (1H, d, J=7.6 Hz), 5.95 (1H, dt,J=1.6, 15.6 Hz), 5.30-5.52 (3H, m), 4.89 (1H, d, J=6.8 Hz), 4.81 (1H, d,J=6.8 Hz), 4.66 (1H, dd, J=3.6, 8.8 Hz), 3.86 (3H, s), 3.71 (1H, dd,J=9.6, 16.4 Hz), 3.45 (3H, s), 3.32 (1H, br d, J=16.4 Hz), 2.61 (1H,dddd, J=1.6, 6.0, 8.8, 15.2 Hz), 2.43 (1H, dddd, J=1.6, 4.4, 8.0, 15.2Hz), 2.26-2.34 (1H, m), 2.08-2.20 (1H, m), 1.75 (1H, dd, J=8.4, 15.2),1.70 (1H, ddd, J=11.6, 11.6, 14.4 Hz), 1.49 (9H, s), 1.43 (1H, dd, J9.2, 15.2 Hz), 0.86 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.5,166.0, 157.0, 143.5, 139.2, 131.5, 130.3, 128.8, 125.5, 124.5, 122.8,109.5, 97.3, 80.4, 79.6, 73.4, 55.9, 55.8, 39.2, 37.93, 37.88, 35.9,34.4, 28.4, 13.5. HRMS Calcd for C₂₇H₃₈O₇Li (MLi⁺): 481.2778. Found:481.2774. 334 a (Mixture of E/Z isomers, ˜3.5-4:1): IR 2954, 1725, 1585,1584, 1469, 1440, 1275, 1249, 1117, 1041 cm⁻¹; ¹H NMR (400 MHz, CDCl₃,peaks corresponding to major isomer) δ 7.24 (1H, app.t, J=7.6 Hz), 7.12(1H, ddd, J=5.6, 8.4, 15.6 Hz), 6.82 (1H, d, J=8.4 Hz), 6.75 (1H, d,J=7.6 Hz), 5.92 (1H, br d, J=15.6 Hz), 5.48 (1H, br dd, J=10.8, 15.2Hz), 5.38 (1H, ddd, J=4.0, 8.8, 8.8 Hz), 5.36 (1H, br dd, J=9.2, 15.2Hz), 4.89 (1H, d, J=6.8 Hz), 4.81 (1H, d, J=6.8 Hz), 4.16 (1H, dd,J=3.6, 8.8 Hz), 3,86 (3H, s), 3.74 (3H, s), 3.71(1H, dd, J=10.8, 16.4Hz), 3.45 (3H, s), 3.32 (1H, dddd, J=2.4, 2.4, 4.8, 16.4 Hz), 2.64 (1H,dddd, J=1.6, 5.6, 8.8, 15.2 Hz), 2.46 (1H, dddd, J=1.2, 4.0, 8.4, 15.2Hz), 2.31 (1H, br d, J=14.0 Hz), 2.10-2.19 (1H, m), 1.76 (1H, dd, J=8.8,15.6 Hz), 1.71 (1H, app.td, J=11.6, 14.0 Hz), 1.45 (1H, dd, J=9.6, 15.2Hz), 0.86 (3H, d, J=7.2 Hz).

[0293] According to the procedure described below for the deprotectionof allyl ester 334 b, the mixture of methyl esters 334 a (250 mg, 0.505mmol) were deprotected with BBr₃ (2.5 equiv.) to give after separationby FC (35% EtOAc/hexanes) 30 mg of the less polar Z-isomer Z-335 a (0.08mmol, 16%) and 121 mg of the more polar E-isomer E-335 a (0.323 mmol,64%). E-335 a: [α] _(D)=+8.7 (c 1.2, CHCl₃) [ent-E-335 a=−7.0 (c 1.1,CHCl₃]; IR 3404, 3162, 2949, 2914, 1729, 1691, 1589, 1468, 1426, 1297,1268, 1249, 1127, 1031, 966 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.31 (1H,app.t, J=8.4 Hz), 6.98 (1H, app.dt, J=7.6, 15.6 Hz), 6.90 (1H, dd,J=0.8, 8.0 Hz), 6.71 (1H, d, J=7.6 Hz), 5.94 (1H, br d, J=15.6 Hz), 5.6(1H, app.dt, J=5.6, 10.8 Hz), 5.49 (1H, br d, J=16 Hz), 5.02-5.13 (1H,m), 3.74 (3H, s), 3.70-3.78 (1H, m), 3.61(1H, br d, J 8.0 Hz), 3.38 (1H,br d, J=16.4 Hz), 2.56-2.68 (2H, m), 2.32-2.40 (1H, m), 2.03 (1H, dd,J=11.2, 15.2 Hz), 1.79-1.96 (2H, m), 1.50 (1H, s), 1.37 (1H, ddd, J=1.2,8.4, 14.8 Hz), 0.92 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 171.4,166.7, 163.3, 144.4, 142.7, 134.5, 133.4, 126.3, 123.9, 122.2, 117.0,112.9, 74.1, 70.6, 51.4, 39.3, 38.6, 37.5, 35.8, 34.7, 13.9. HRMS Calcdfor C₂₁H₂₆O₆Li (MLi⁺): 381.1889. Found: 381.1889. Ent-Z-335 a: [α]_(D)=−40.8 (c 1.2, CHCl₃); IR 3200-3600, 2955, 2926, 1722, 1652, 1606,1588, 1464, 1441, 1293, 1248, 1214, 1176, 1119, 1064, 1028, 760 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 7.31 (1H, app.t, J=8.0 Hz), 6.90 (1H, d, J=8.0Hz), 6.70 (1H, d, J=7.2 Hz), 6.30 (1H, app.dt, J=7.2, 11.2 Hz), 5.92(1H, d, J=11.2 Hz), 5.66 (1H, app.dt, J=6.0, 11.2 Hz), 5.51 (1H, br d,J=16 Hz), 5.02-5.12 (1H, m), 3.78 (1H, dd, J=6.4, 16.8), 3.69 (3H, s),3.59(1H, dd, J=3.2, 8.8 Hz), 3.36 (1H, d, J=16.8 Hz), 3.15-3.24 (1H, m),3.00-3.08 (1H, m), 2.32-2.40 (1H, m), 2.08 (1H, dd, J=10.8, 14.8 Hz),1.79-1.96 (2H, m), 1.40 (1H, dd, J=7.6, 14.8 Hz), 0.93 (3H, d, J=6.8Hz).

[0294] HRMS Calcd for C₂₁H₂₆O₆Li (MLi⁺): 381.1889. Found: 381.1891.

[0295] To a solution of allyl ester 334 b (343 mg, 0.748 mmol) in CH₂Cl₂(40 mL) was added at −78° C. BBr₃ (0.29 mL, 1.87 mmol). After stirringfor 20 min, a saturated aqueous solution of NaHCO₃ (10 mL) and H₂O (100mL) was added followed by extraction with EtOAc (3×), drying (MgSO₄) andconcentration. The residue was purified by FC (25% EtOAc/hexanes) toyield 243 mg of diol 335 b (0.607 mmol, 81%). 335 b: [α] _(D)=4.3 (c0.88, CHCl₃); IR 3407, 3172, 2959, 1731, 1690, 1656, 1590 cm⁻¹; ¹H NMR(400 MHz, CDCl₃) δ 11.0 (1H, s), 7.31 (1H, dd, J=7.6, 8.4 Hz), 7.01 (1H,app.dt, J=7.2, 15.6 Hz), 6.89 (1H, dd, J=0.8, 8.4 Hz), 6.71 (1H, dd,J=0.8, 7.6 Hz), 5.97 (1H, d, J=15.6 Hz), 5.95 (1H, ddt, J=5.6, 10.4,15.6 Hz), 5.64 (1H, app.dt, J=5.2, 11.2 Hz), 5.44-5.52 (1H, m), 5.33(1H, app.qd, J=1.2, 15.6 Hz), 5.24 (1H, app.qd, J=1.2, 10.4 Hz),5.02-5.13 (1H, m), 4.65 (1H, app.td, J=1.2, 5.6 Hz), 3.74 (1H, dd,J=5.6, 16.4 Hz), 3.62 (1H, dd, J=3.2, 8.8 Hz), 3.38 (1H, br d, J=16.4Hz), 2.56-2.69 (2H, m), 2.31-2.40 (1H, m), 2.03 (1H, dd, J=11.2, 15.2),1.79-1.97 (2H, m), 1.52-1.72 (1H, m), 1.44 (1H, ddd, J=1.2, 8.4, 14.8Hz), 0.93 (3H, d, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 171.1, 165.9,163.1, 143.7, 142.5, 134.5, 133.2, 132.3, 126.5, 124.5, 123.9, 118.5,116.9, 113.1, 73.2, 70.5, 65.3, 39.3, 38.6, 38.0, 37.5, 35.6, 13.9; MS(CI) m/z (%): 401 (28), 383 (30), 343 (37), 325 (100); HRMS (FAB) Calcdfor C₂₃H₂₉O₆ (MH⁺): 401.1964. Found: 401.1973.

[0296] To a solution of 335 b (208 mg, 0.52 mmol), imidazole (250 mg,3.63 mmol) and DMAP (25 mg) in DMF (10 mL) was addedtert.butyldimethylsilyl chloride (479 mg, 3.12 mmol). After stirring for10 h at rt, the mixture was poured into water (100 mL), extracted withEt₂O (3×), and the combined organics washed with brine, dried over MgSO₄and concentrated in vacuo. FC (2% EtOAc/hexanes) yields 301 mg ofsilylether 336 (0.48 mmol, 92%). 336: [α]_(D)=−1.0 (c 0.5, CHC₃); IR ¹HNMR (400 MHz, CDCl₃) δ 7.12 (1H, app.t, J=8.0 Hz), 6.93 (1H, app.dt,J=7.2, 15.6 Hz), 6.75 (1H, d, J=7.6 Hz), 6.72 (1H, d, J=8.0 Hz), 5.95(1H, ddt, J=6.0, 10.4, 17.2 Hz), 5.94 (1H, d, J=15.6 Hz), 5.29-5.46 (3H,m), 5.33 (1H, app.qd, J=1.6, 17.2 Hz), 5.24 (1H, app.qd, J=1.6, 10.4Hz), 4.64 (2H, dd, J=1.6, 5.6 Hz), 4.26 (1H, dd, J=3.2, 8.8 Hz), 3.65(1H, dd, J=8.8, 16.4 Hz), 3.32 (1H, br d, J=16.4 Hz), 2.55-2.60 (2H, m),2.22-2.30 (1H, m), 1.76-1.85 (1H, m), 1.65-1.75 (1H, m), 1.67 (1H, dd,J=8.8, 15.6 Hz), 1.41 (1H, dd, J=8.8, 15.6 Hz), 0.96 (9H, s), 0.90 (9H,s), 0.83 (3H, d, J=6.4 Hz), 0.22 (3H, s), 0.20 (3H, s), 0.15 (3H, s),0.12 (3H, s); HRMS (FAB) Calcd for C₃₅H₅₇O₆Si₂ (MH⁺): 629.3694.

[0297] Found: 629.3687.

[0298] To a solution of morpholine (0.41 mL, 4.78 mmol) and allyl ester336 (301 mg, 0.478 mmol) in THF (10 mL) was added Pd(PPh₃)₄ (28 mg,0.024 mmol). The mixture was stirred at rt in the dark for 4 h and brine(80 mL) was added. The aqueous phase was extracted with EtOAc (4×) andthe combined organic layers were dried (MgSO₄) and concentrated invacuo. The residue was purified by FC (20% EtOAc/hexanes) to give 270 mgof acid 337 (0.458 mmol, 96%). 337: [α]_(D)=+2.0 (c 1.84, CHCl₃); IR2956, 2930, 2858, 1728, 1700, 1652, 1582, 1457 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 7.13 (1H, app.t, J=8.0 Hz), 7.02 (1H, app.dt, J=7.2, 15.6 Hz),6.75 (1H, d, J=7.6 Hz), 6.72 (1H, d, J=8.0 Hz), 5.93 91H, d, J=15.6 Hz),5.30-5.46 (3H, m), 4.26 (1H, dd, J=3.2, 8.8 Hz), 3.66 (1H, dd, J=8.8,16.0 Hz), 3.32 (1H, br d, J=16.0 Hz), 2.60 (2H, app.t, J=6.8 Hz),2.21-2.30 (1H, m), 1.75-1.86 (1H, m), 1.35-1.45 (1H, m), 0.96 (9H, s),0.91 (9H, s), 0.83 (3H, d, J=6.4 Hz), 0.22 (3H, s), 0.20 (3H, s), 0.15(3H, s), 0.12 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 171.5, 168.4, 152.9,146.5, 138.9, 131.6, 129.8, 128.5, 127.5, 123.8, 123.4, 118.0, 72.5,72.2, 38.7, 38.3, 38.2, 37.3, 36.5, 26.1, 25.9, 18.5, 18.2, 13.2, −3.9,−4.19, −4.24, −4.3; MS (ES) m/z (%): 589 (10), 531 (21), 457 (18), 367(40), 115 (78), 73 (100); HRMS (FAB) Calcd for C₃₂H₅₃O₆Si₂ (MH⁺):589.3381. Found: 589.3391.

[0299] According to the procedure described for the synthesis of allylester 334 b, aldehyde 357 (22 mg, 0.058 mmol) gave 25 mg of allyl estervii (0.054 mmol, 93%). Allyl ester vii: [α_(D)=−54.2 (c 1.1, CHCl₃); IR2933, 1724, 1471 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.25 (1H, t, J=8.0 Hz),7.08 (1H, ddd, J=6.8, 7.6, 16.0 Hz), 6.77 (2H (1H +1H), d, J=8.0 Hz),6.72 (1H, d, J=8.0 Hz), 5.85-6.0 (1H, m), 5.96 (1H, d, J=16.0 Hz),5.36-5.45 (1H, m), 5.33 (1H, br.d, J=17.2 Hz), 5.24 (1H, br.d, J=10.4Hz), 4.80 (1H, d, J=6.8 Hz), 4.75 (1H, d, J=6.8 Hz), 4.64 (2H, br.d,J=5.2 Hz), 3.83 (3H, s), 3.75-3.81 (1H, m), 3.43 (3H, s), 2.86 (1H, ddd,J=5.2, 12.8, 12.8 Hz), 2.75 (1H, dddd, J=1.6, 6.4, 8.4, 14.8 Hz), 2.56(1H, dddd, J=1.2, 4.4, 7.6, 14.8 Hz), 2.47 (2H, ddd, J=5.2, 12.4, 12.8Hz), 1.90-2.06 (2H, m), 1.60-1.74 (3H, m), 1.39-1.56 (2H, m), 1.14-1.32(2H, m), 0.89 (3H, d, J=6.4 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 167.9, 166.0,156.7, 144.5, 141.4, 132.4, 130.7, 123.8, 123.6, 122.1, 118.3, 108.9,97.1, 72.4, 65.1, 55.9, 38.6, 35.4, 33.6, 31.9, 26.4, 15.2; HRMS (FAB)Calcd for C₂₆H₃₆O₇Li (MLi⁺): 467.2621. Found: 467.2621.

[0300] According to the procedure described for the synthesis of 335 b,deprotection of allyl ester vii (25 mg, 0.054 mmol) with BBr₃ (2.5equiv.) gave 21 mg of 358 (0.052 mmol, 97%). 358: [α]_(D)=−21.2 (c 0.61,CHCl₃); IR 3448, 2935, 1722, 1652, 1606, 1449 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 10.95 (1H, s), 7.29 (1H, t, J=8.0 Hz), 6.93 (1H, ddd, J=7.6,8.0, 15.2 Hz), 6.84 (1H, d, J=8.0 Hz), 6.72 (1H, d, J=8.0 Hz), 5.94 (1H,ddt, J=5.6, 10.4, 17.2 Hz), 5.91 (1H, br.d (lr), J=15.2 Hz), 5.32 (1H,ddt, J=1.2, 1.6, 17.2 Hz), 5.24 (1H, br.d, J=10.4 Hz), 5.22-5.30 (1H,m), 4.64 (2H, br.d, J=5.6 Hz), 3.89-3.95 (1H, m), 3.68-3.76 (1H, m),2.64 (2H, ddd, J=1.2, 8.0, 8.0 Hz), 2.16-2.27 (2H, m), 1.66-1.80 (3H,m), 1.50-1.64 (4H, m), 1.32-1.44 (1H, m), 1.15-1.26 (1H, m), 0.96 (3H,d, J=6.6 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 171.8, 165.7, 163.0, 146.7,142.9, 134.7, 132.3, 124.9, 122.8, 118.5, 116.1, 111.7, 74.8, 73.6,65.4, 38.3, 36.8, 33.7, 31.6, 25.3, 25.0, 16.3; HRMS (FAB) Calcd forC₂₃H₃₀O₆Li (MLi⁺): 409.2202. Found: 409.2202.

[0301] According to the procedure described for the synthesis ofbis-silyl ether 336, silylation of 358 (20 mg, 0.050 mmol) provided 21mg of bis-silyl ether 359 (0.033 mmol, 67%). 359: ¹H NMR (400 MHz,CDCl₃) δ 7.14 (1H, dd, J=7.6, 8.0 Hz), 6.94 (1H, ddd, J=6.8, 7.6, 15.6Hz), 6.75 (1H, d, J=7.6 Hz), 6.69 (1H, d, J=8.0 Hz), 5.95 (1H, br.d(lr), J=15.6 Hz), 5.94 (1H, ddt, J=5.6, 10.4, 17.2 Hz), 5.33 (1H, ddt(lr), J=1.2, 1.6, 17.2 Hz), 5.30-5.38 (1H, m), 5.24 (1H, ddt (lr),J=0.8, 1.2, 10.4 Hz), 4.64 (2H, ddd (lr), J=0.8, 1.6, 5.6 Hz), 3.79-3.86(1H, m), 2.98-3.10 (1H, m), 2.51-2.64 (2H, m), 2.20-2.30 (1H, m),1.96-2.06 (1H, m), 1.50-1.67 (5H, m), 1.23-1.48 (3H, m), 0.97 (9H, s),0.89 (9H, s), 0.88 (3H, d, J=6.8 Hz), 0.25 (3H, s), 0.22 (3H, s), 0.09(3H, s), 0.05 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 171. HRMS Calcd forC₃₅H₅₈O₆Si₂Li (MLi⁺): 637.3932. Found: 637.3925.

[0302] According to the procedure described for the synthesis ofcarboxylic acid 337, allyl ester 359 (21 mg, 0.033 mmol) was deprotectedto provide 18 mg of carboxylic acid viii (0.030 mmol, 92%). viii:[α]_(D)=−24.7 (c 0.85, CHCl₃); IR 2930, 2858, 1728, 1701, 1657, 1464,1253 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 7.15 (1H, dd, J=7.6, 8.0 Hz), 7.03(1H, ddd, J=6.8, 7.6, 16.0 Hz), 6.75 (1H, d, J=7.6 Hz), 6.70 (1H, d,J=8.0 Hz), 5.94 (1H, br.d (lr), J=16.0 Hz), 5.32-5.41 (1H, m), 3.80-3.88(1H, m), 2.98-3.12 (1H, m), 2.55-2.68 (2H, m), 2.20-2.30 (1H, m),1.96-2.08 (1H, m), 1.50-1.70 (5H, m), 1.20-1.48 (3H, m), 0.97 (9H, s),0.89 (9H, s), 0.88 (3H, d, J=6.8 Hz), 0.25 (3H, s), 0.22 (3H, s), 0.11(3H, s), 0.05 (3H, s), 0.05 (3H, s); ¹³C NMR (75 MHz, CDCl₃) δ 171.1,167.2, 153.5, 146.2, 142.4, 130.6, 125.5, 124.1, 122.7, 117.0, 77.4,71.0, 39.1, 37.7, 33.2, 30.6, 26.1, 25.9, 18.4, 18.3, 16.3, −3.9, −4.11,−4.13, −4.4; MS (EI) m/z (%): 533 (M⁺−57, 8), 459 (7), 367 (100), 365(66), 309 (12), 115 (64), 75 (24), 73 (52), 57 (83). HRMS Calcd forC₃₂H₅₄O₆Si₂Li (MLi⁺): 597.3619. Found: 597.3617.

[0303] To a stirred solution of acid 337 (52 mg, 0.0883 mmol) and(PhO)₂P(O)N₃ (77.3 μL; 0.353 mmol) in benzene (4 mL) was added Et₃N (58μL) at RT. After stirring for 14 h at RT, the solvent was removed andthe residue was purified by FC (silicagel, 2.5% EtOAc in hexanes). Thecorresponding acyl azide 338 was obtained in 92% yield (50 mg). 338: ¹HNMR (400 MHz, CDCl₃) δ 7.13 (1H, dd, J=7.6, 8.0 Hz), 7.00 (1H, app.dt,J=7.2, 15.6 Hz), 6.75 (1H, d, J=7.6 Hz), 6.72 (1H, d, J=8.0 Hz), 5.92(1H, d, J=15.6 Hz), 5.29-5.46 (3H, m), 4.26 (1H, dd, J=3.6, 8.4 Hz),3.65 (1H, dd, J=9.2, 16.4 Hz), 3.33 (1H, br d, J=16.4 Hz), 2.54-2.65(2H, m), 2.27 (1H, br.d, J=14.0 Hz), 1.75-1.86 (1H, m), 1.65-1.74 (1H,m), 1,67 (1H, dd, J=8.8, 15.2 Hz), 1.37 (1H, dd, J=8.8, 15.2 Hz), 0.96(9H, s), 0.92 (9H, s), 0.83 (3H, d, J=6.4 Hz), 0.22 (3H, s), 0.20 (3H,s), 0.14 (3H, s), 0.12 (3H, s).

[0304] Acyl azide ix was prepared in an identical manner as describedfor the preparation of acyl azide 338. ix: ¹H NMR (400 MHz, CDCl₃) δ7.15 (1H, dd, J=7.6, 8.0 Hz), 7.01 (1H, ddd, J=7.6, 7.2, 15.6 Hz), 6.76(1H, d, J=7.6 Hz), 6.70 (1H, d, J=8.0 Hz), 5.92 (1H, br.d (lr), J=15.6Hz), 5.32-5.40 (1H, m), 3.80-3.88 (1H, m), 2.98-3.10 (1H, m), 2.53-2.68(2H, m), 2.21-2.30 (1H, m), 1.96-2.06 (1H, m), 1.50-1.68 (5H, m),1.23-1.45 (3H, m), 0.97 (9H, s), 0.90 (9H, s), 0.87 (3H, d, J=6.8 Hz),0.25 (3H, s), 0.22 (3H, s), 0.11 (3H, s), 0.06 (3H, s).

[0305] Acyl azide 338 (12 mg; 0.0195 mmol) in benzene (1 mL) was stirredat 75° C. for 6 h, after which the solvent was removed and the residue(isocyanate 339) dissolved in diethyl ether (1 mL). In a separate flask,a 0.15 M solution of 1-lithio-1,3-hexadiene (1:1 mixture of 1Z,3Z and1Z,3E isomers) was prepared by the addition of t-BuLi (2.05 equiv withrespect to the bromide) to a solution of the corresponding bromide inTHF at −78° C. After stirring for 45 min at −78° C. and warming to RT,the organolithium (0.15 M in THF; 143 μL; 0.0215 mmol) was addeddropwise to the ethereal solution of isocyanate 339 at −78° C. Themixture was allowed to reach 0° C. over a 1 h period followed by theaddition of pH 7.0 phosphate buffer. Extraction with diethyl ether (3×),drying (Na₂SO₄), concentration and rapid purification by FC (silicagel,6% EtOAc in hexanes containing 0.2% Et₃N) gave 3.7 mg of a less polarproduct 342 and 5.9 mg of a more polar product 341.

[0306] The less polar product 342 was treated with 250 μL of a solutionprepared from 0.5 g commercial HF-pyridine in 1.25 mL pyridine and 6.75mL THF. The more polar product 341 was similarly treated with 410 μL ofthe same solution. After stirring for 48 h at RT, the reactions werequenched with a phosphate buffer (pH 7.0; 10 mL), extracted with EtOAc(4×), dried (Na₂SO₄) and concentrated. The product derived fromdeprotection of 341 was purified by normal-phased semi-preparative HPLC(5μ Luna silicagel; 250×10 mm column; 25% acetone in hexanes, t_(R)=25min) yielding 1.7 mg of a 1:1 mixture of salicylihalamide A (301 a) andthe corresponding geometrical isomer 301 c (20% from acyl azide 338).The product derived from deprotection of 342 provided two fractionsafter HPLC purification (35% acetone in hexanes): 1.5 mg of 343(t_(R)=25.7 min; 10% yield from acyl azide 338) and 1.5 mg of 344(t_(R)=26.7 min; 10% yield from acyl azide 338). The combined overallyield for 301 a,c and 343-344 from acyl azide 338 is 40%. 343: ¹H NMR(400 MHz, CD₃OD) δ 7.14 (1H, app.t, J=8.0 Hz), 7.13 (1H, app.t, J=8.0Hz), 7.06 (1H, app.t, J=11.2 Hz), 6.75 (1H, d, J=8.0 Hz), 6.72 (1H, d,J=14.0 Hz), 6.71 (1H, d, J=8.0 Hz), 6.68 (1H, d, J=7.6 Hz), 6.67 (1H, d,J=7.6 Hz), 6.61 (1H, app.t, J=12.0 Hz), 6.44 (1H, d, J=14.0 Hz), 6.09(1H, d, J=11.6 Hz), 5.81-5.90 (2H, m), 5.51 (1H, ddd, J=6.4, 8.4, 14.8Hz), 5.23-5.48 (6H, m), 4.18 (1H, dd, J=3.6, 9.6 Hz), 4.17 (1H, dd,J=3.6, 9.6 Hz), 3.62 (1H, dd, J=8.8, 15.6 Hz), 3.58 (1H, dd, J=8.8, 15.6Hz), 3.37 (2H, br d, J=15.6 Hz), 2.42-2.61 (3H, m), 2.25-2.40 (3H, m),2.15-2.24 (2H, m), 1.84-1.95 (2H, m), 1.68-1.87 (4H, m), 1.30-1.43 (2H,m), 0.97 (3H, t, J=7.2 Hz), 0.87 (6H, d, J=6.8 Hz); MS (ES) m/z (%):819.33 ([M+Na]⁺, 35), 797.34 ([M+H]⁺, 100); HRMS (FAB) Calcd forC₄₆H₅₇N₂O10 (MH⁺): 797.4013. Found: 797.4015. 344: ¹H NMR (400 MHz,CD₃OD) δ 7.21 (1H, dd, J=11.2, 15.2 Hz), 7.14 (1H, app.t, J=8.0 Hz),7.13 (1H, app.t, J=7.6 Hz), 6.75 (1H, d, J=7.6 Hz), 6.74 (1H, d, J=13.6Hz), 6.71 (1H, d, J=8.0 Hz), 6.68 (1H, d, J=7.2 Hz), 6.67 (1H, d, J=7.6Hz), 6.45 (1H, d, J=13.6 Hz), 6.29 (1H, app.t, J=11.2 Hz), 6.07 (1H, dt,J=6.8, 15.2 Hz), 5.99 (1H, d, J=11.6 Hz), 5.84 (1H, app.dt, J=6.8, 14.0Hz), 5.51 (1H, ddd, J=6.4, 8.0, 14.4 Hz), 5.23-5.47 (6H, m), 4.18 (1H,dd, J=4.4, 8.8 Hz), 4.17 (1H, dd, J=4.4, 8.8 Hz), 3.61 (1H, dd, J=8.8,16.8 Hz), 3.58 (1H, dd, J=8.4, 16.4 Hz), 3.37 (2H, br d, J=16.4 Hz),2.42-2.55 (3H, m), 2.25-2.40 (3H, m), 2.15-2.24 (2H, m), 1.84-1.95 (2H,m), 1.69-1.87 (4H, m), 1.32-1.43 (2H, m), 1.04 (3H, t, J=7.2 Hz), 0.87(6H, d, J=6.8 Hz); MS (ES) m/z (%): 819.30 ([M+Na]⁺, 60), 797.34([M+H]⁺, 100); HRMS (FAB) Calcd for C₄₆H₅₇N₂O₁₀ (MH⁺): 797.4013. Found:797.4021.

[0307] Following the procedure described above, acyl azide 338 (9.0 mg;0.0146 mmol) was converted to isocyanate 339, followed by the additionof n-hexyllithium (0.15 M in THF; prepared from the bromide as describedabove). Workup and rapid purification by FC (silicagel, 15% EtOAc inhexanes containing 0.2% Et₃N) gave 4.0 mg of a less polar product x and4.2 mg of a more polar product xi. Deprotection and purification bysemi-preparative HPLC as described above yielded 1.4 mg of 346 (22%) and1.6 mg of the corresponding dimer 345 (14%), respectively. 346: ¹H NMR(400 MHz, CD₃OD) δ 7.15 (1H, app.t, J=8.0 Hz), 6.76 (1H, d, J=14.0 Hz),6.74 (1H, d, J=8.0 Hz), 6.67 (1H, d, J=7.2 Hz), 5.25-5.44 (4H, m), 4.13(1H, dd, J=3.2, 8.8 Hz), 3.57 (1H, dd, J=8.0, 16.4 Hz), 3.37 (1H, br d,J=16.4 Hz), 2.25-2.46 (3H, m), 2.22 (2H, t, J 7.2 Hz), 1.84-1.95 (1H,m), 1.71-1.83 (2H, m), 1.57-1.66 (2H, m), 1.28-1.42 (7H, m), 0.91 (3H,t, J=7.2 Hz), 0.87 (3H, d, J=6.8 Hz); MS (ES) m/z (%): 466.24 ([M+Na]⁺,17), 444.26 ([M+H]⁺, 100); HRMS (FAB) Calcd for C₂₆H₃₈NO₅ (MH⁺):444.2750. Found: 444.2759. 345: ¹H NMR (400 MHz, CD₃OD) δ 7.14 (1H,app.t, J=7.6 Hz), 7.12 (1H, app.t, J=7.2 Hz), 6.75 (1H, d, J=8.4 Hz),6.73 (1H, d, J=14.4 Hz), 6.72 (1H, d, J=8.8 Hz), 6.67 (2H, d, J=7.2 Hz),6.35 (1H, d, J=14.0 Hz), 6.00 (1H, app.dt, J=7.6, 14.4 Hz), 5.50 (1H,ddd, J=6.4, 8.4, 14.4 Hz), 5.25-5.46 (6H, m), 4.19 (1H, dd, J 3.2, 9.2Hz), 4.16 (1H, dd, J=3.2, 8.8 Hz), 3.60 (1H, dd, J=8.0, 16.0 Hz), 3.59(1H, dd, J=7.6, 16.0 Hz), 3.37 (2H, br d, J=16.0 Hz), 2.63 (1H, app.dt,J=7.6, 16.8 Hz), 2.40-2.56 (4H, m), 2.24-2.39 (3H, m), 1.70-1.95 (6H,m), 1.47-1.57 (2H, m), 1.21-1.43 (8H, m), 0.87 (3H, d, J=6.8 Hz), 0.86(3H, d, J=6.8 Hz), 0.86 (3H, t, J=7.2 Hz); MS (ES) m/z (%): 823.33([M+Na]⁺, 50), 801.37 ([M+H]⁺, 100); HRMS (FAB) Calcd for C₄₆H₆₁N₂O₁₀(MH⁺): 801.4326. Found: 801.4334.

[0308] A solution of acyl azide 338 (10.0 mg; 0.0163 mmol) and1-pentanol (11 μL; 0.1019 mmol) in benzene (1 mL) was stirred for 8 h at80° C. After removal of the solvent, the residue was treated with 780 μLof a solution prepared from 0.5 g commercial HF-pyridine in 1.25 mLpyridine and 6.75 mL THF. After stirring for 24 h at RT, the reactionwere quenched with a phosphate buffer (pH 7.0; 10 mL), extracted withEtOAc (4×), dried (Na₂SO₄) and concentrated. Purification by FC(silicagel; 25% acetone in hexanes) yielded 3.4 mg of carbamate 351(0.00763 mmol; 47%). 351: [α]_(D)=−18.8 (c 0.17, MeOH); ¹H NMR (400 MHz,CD₃OD) δ 7.14 (1H, app.t, J=8.0 Hz), 6.73 (1H, d, J=8.0 Hz), 6.67 (1H,d, J=7.2 Hz), 6.49 (1H, d, J=14.4 Hz), 5.26-5.44 (3H, m), 5.18 (1H,app.dt, J=7.2, 14.4 Hz), 4.13 (1H, dd, J=3.6, 9.2 Hz), 4.07 (2H, t, 6.4Hz), 3.57 (1H, dd, J=8.4, 16.4 Hz), 3.36 (1H, br d, J=16.4 Hz),2.25-2.44 (3H, m), 1.84-1.95 (1H, m), 1.71-1.82 (2H, m), 1.60-1.69 (2H,m), 1.33-1.41 (5H, m), 0.93 (3H, t, J=7.6 Hz), 0.87 (3H, d, J=6.4 Hz);¹³C NMR (75 MHz, CD₃OD) δ 171.2, 157.2, 156.6, 140.8, 131.83, 131.76,127.8, 123.2, 122.6, 115.4, 106.9, 76.4, 72.1, 66.4, 39.1, 38.9, 38.7,37.5, 36.6, 29.9, 29.3, 23.5, 14.5, 13.7; MS (ES) m/z (%): 468.20([M+Na]⁺, 26), 446.23 ([M+H]⁺, 100); HRMS (FAB) Calcd for C₂₅H₃₆NO₆(MH⁺): 446.2543. Found: 446.2528.

[0309] According to the procedure described for the synthesis ofcarbamate 351, acyl azide ix (15 mg, 0.02435 mmol) gave 4.7 mg ofcarbamate 361. 361: [α]_(D)=−13.2 (c 0.24, MeOH); IR 3387, 2933, 1727,1710, 1680, 1603, 1293, 1243, 1113 cm⁻¹; ¹H NMR (400 MHz, CD₃OD) δ 7.25(1H, app.t, J=7.6 Hz), 6.75 (1H, d, J=8.4 Hz), 6.73 (1H, d, J=7.6 Hz),6.44 (1H, d, J=14.0 Hz), 5.14-5.22 (1H, m), 5.05 (1H, app.dt, J=7.6,14.0 Hz), 4.04 (2H, t, J=6.4 Hz), 3.82-3.88 (1H, m), 3.66 (1H, ddd, 3.6,12.4, 12.4 Hz), 2.39 (2H, app.t, J=6.6 Hz), 2.21 (1H, ddd, J=6.4, 12.4,12.4 Hz), 2.10-2.20 (1H, m), 1.40-1.80 (9H, m), 1.30-1.40 (5H, m), 0.95(3H, d, J=6.8 Hz), 0.92 (3H, t, J=6.4 Hz); ¹³C NMR (75 MHz, CD₃OD) δ172.5, 162.3, 156.6, 147.0, 134.8, 128.5, 123.4, 116.3, 115.2, 105.5,76.3, 66.4, 37.4, 34.4, 32.7, 29.9, 29.3, 23.5, 16.6, 14.5; MS (ES) m/z448.20 ([M+H]⁺, 100), 470.19 ([M+Na]⁺, 32). HRMS Calcd for C₂₅H₃₇NO₆Li(MLi⁺): 454.2781. Found: 454.2779.

[0310] To a solution of carbamate 361 (1 mg, 0.00268 mmol) in MeOH (1mL) was added 10% Pd/C (1 mg) and the resulting suspension was stirredunder an atmosphere of hydrogen gas (balloon) at rt for 16 h. Thecatalyst was filtered and the filtrate concentrated and purified by HPLC(20% acetone/hexanes) to yield 0.3 mg of carbamate 362 (0.000667 mmol,25%). 362: ¹H NMR (400 MHz, CD₃OD) δ 7.24 (1H, app.t, J=8.0 Hz), 6.75(1H, d, J=8.4 Hz), 6.73 (1H, d, J=7.2 Hz), 5.24-5.32 (1H, m), 3.99 (2H,t, J=6.8 Hz), 3.86-3.92 (1H, m), 3.55 (1H, ddd, 3.6, 12.4, 12.4 Hz),3.11 (2H, app.t, J=6.8 Hz), 2.11-2.30 (2H, m), 1.45-1.77 (13H, m),1.28-1.38 (5H, m), 0.94 (3H, d, J=6.8 Hz), 0.91 (3H, t, J=7.6 Hz); MS(ES) m/z 450.24 ([M+H]⁺, 100), 472.24 ([M+Na]⁺, 67). HRMS Calcd forC₂₅H₃₉NO₆Li (MLi⁺): 456.2937. Found: 456.2935.

[0311] To a solution of 331 (6.4 mg, 0.0169 mmol), octanoic acid (5.3μL, 0.034 mmol) and PPh₃ (8.8 mg, 0.034 mmol) in Et₂O (1 mL) was addeddiethylazodicarboxylate (DEAD, 5.5 μL, 0.034 mmol). After stirring for1.5 h at rt, H₂O (5 mL) was added and an extraction was performed withEt₂O (3×). The combined organic layers were dried (MgSO₄) andconcentrated in vacuo. The residue was purified by FC (10%EtOAc/hexanes) to give 6.7 mg of octanoate ester xii (0.0133 mmol, 79%).xii: ¹H NMR (400 MHz, CDCl₃) δ 7.23 (1H, app.t, J=8.4 Hz), 6.80 (1H, d,J=8.4 Hz), 6.75 (1H, d, J=7.6 Hz), 5.42-5.53 (2H, m), 5.35 (1H, app.ddt,J=2.4, 9.6, 15.2 Hz), 4.90 (1H, d, J=6.8 Hz), 4.82 (1H, d, J=6.8), 4.27(1H, ddd, J=4.8, 7.6, 11.2 Hz), 4.22 (1H, ddd, J=6.4, 8.8, 11.2 Hz),4.18 (1H, dd, J=3.6, 9.2 Hz), 3.18 (3H, s), 3.72 (1H, dd, J=9.6, 16.4Hz), 3.47 (3H, s), 3.32 (1H, dddd, J=2.0, 2.0, 4.4, 16.4 Hz), 2.27-2.36(1H, m), 2.29 (2H, t, J=7.2 Hz), 2.07-2.18 (1H, m), 1.87-2.06 (2H, m),1.78 (1H, dd, J=8.8, 15.6 Hz), 1.71 (1H, app.dt, J=11.6, 14.4 Hz),1.53-1.67 (2H, m), 1.43 (1H, dd, J=9.2, 15.2 Hz), 1.22-1.34 (8H, m),0.87 (3H, d, J=6.8 Hz), 0.87 (3H, t, J=6.8 Hz); ¹³C NMR (75 MHz, CDCl₃)δ 174.2, 168.5, 156.7, 139.2, 131.6, 130.3, 128.7, 124.5, 122.9, 109.3,97.1, 79.4, 71.5, 61.1, 55.8, 55.6, 38.0, 37.9, 36.1, 35.4, 34.6, 34.2,31.9, 29.3, 29.1, 25.2, 22.8, 14.3, 13.5. HRMS Calcd for C₂₉H₄₄O₇Li(MLi⁺): 511.3247. Found: 511.3251. The bis-MOM derivative xiv wasprepared similarly from alcohol xiii. xiv: [α]_(D)=+47.6 (c 0.63,CHCl₃); IR 2927, 1733, 1587, 1463, 1273, 1157, 1040 cm⁻¹; ¹H NMR (400MHz, CDCl₃) δ 7.21 (1H, dd, J=8.4, 7.6 Hz), 7.09 (1H, d, J=8.4 Hz), 6.80(1H, d, J=7.6 Hz), 5.42-5.54 (2H, m), 5.34 (1H, br.dd, K=9.6, 14.8 Hz),5.22 (1H, d, J=6.8 Hz), 5.17 (1H, d, J=6.8), 4.89 (1H, d, J=6.8 Hz),4.82 (1H, d, J=6.8),4.21-4.33 (2H, m), 4.16 (1H, dd, J=3.6, 9.6 Hz),3.72 (1H, dd, J=9.6, 16.4 Hz), 3.47 (3H, s), 3.43 (3H, s), 3.33 (1H,br.d, J=16.4 Hz), 2.28-2.36 (1H, m), 2.28 (2H, t, J=7.6 Hz), 2.09-2.20(1H, m), 1.88-2.08 (2H, m), 1.81 (1H, dd, J=8.4, 16.0 Hz), 1.71 (1H,ddd, J=11.2, 11.2, 14.0 Hz), 1.52-1.64 (2H, m), 1.44 (1H, dd, J=9.6,16.0 Hz), 1.20-1.34 (8H, m), 0.87 (3H, d, J=6.8 Hz), 0.87 (3H, t, J=6.4Hz); ¹³C NMR (75 MHz, CDCl₃) δ 174.2, 168.3, 154.5, 139.3, 131.6, 130.3,128.7, 125.3, 124.0, 113.0, 97.1, 94.6, 79.6, 71.6, 61.1, 56.2, 55.8,37.95, 37.92, 35.9, 35.4, 34.5, 34.2, 31.9, 29.3, 29.1, 25.2, 22.8,14.3, 13.5. HRMS Calcd for C₃₀H₄₆O₈Li (MLi⁺): 541.3353. Found: 541.3361.

[0312] According to the procedure described for the deprotection of 334,deprotection of octanoate xii (9 mg, 0.0178 mmol) with BBr₃ (2.5 equiv.)yielded 5 mg of octanoate 355 (0.011 mmol, 63%). 355: [α]_(D)=−3.72 (c0.2, CHCl₃); IR 3412, 3152, 2918, 2850, 1738, 1686, 1591, 1467 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 11.0 (1H, br s), 7.30 (1H, dd, J=7.6, 8.4 Hz),6.90 (1H, dd, J=0.8, 8.4 Hz), 6.71 (1H, dd, J=0.8, 7.6 Hz), 5.62 (1H,app.ddt, J=0.8, 6.0, 12.0 Hz), 5.49 (1H, br d, J=15.6 Hz), 5.03-5.13(1H, m), 4.24 (1H, app.dt, J=6.0, 6.4, 11.6 Hz),4.18 (1H, app.dt, J=6.0,11.6 Hz), 3.76(1H, dd, J=6.0, 17.2 Hz),3.64 (1H, dd, J=3.6, 8.8 Hz),3.39 (1H, br d, J=17.2 Hz), 2.30-2.40 (1H, m), 2.27 (2H, t, J=7.2 Hz),1.99-2.09 (3H, m), 1.78-1.97 (2H, m), 1.54-1.64 (2H, m), 1.39 (1H, ddd,J=0.8, 8.8, 15.2 Hz), 1.20-1.34 (8H, m), 0.93 (3H, d, J=6.8 Hz), 0.88(3H, t, J=7.2 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 174.2, 171.2, 162.8, 142.4,134.3, 133.1, 126.6, 123.8, 117.0, 113.4, 72.3, 70.6, 60.8, 39.3, 38.6,37.5, 34.7, 34.5, 31.9, 29.9, 29.3, 29.1, 25.1, 22.8, 14.3, 13.9; MS(CI) m/z (%): 447 (30), 429 (70), 303 (36), 285 (75), 145 (100); HRMS(FAB) Calcd for C₂₆H₃₈O₆ (MH⁺): 446.2668. Found: 446.2658.

[0313] To a solution of xiii (25 mg, 0.061 mmol), p-bromo benzoic acid(49 mg, 0.245 mmol) and PPh₃ (65 mg, 0.245 mmol) in Et₂O (6 mL) wasadded diethylazodicarboxylate (DEAD, 40 μL, 0.245 mmol). After stirringfor 2.5 h at rt, H₂O (10 mL) was added and an extraction was performedwith Et₂O (3×). The combined organic layers were dried (MgSO₄) andconcentrated in vacuo. The residue was purified by FC (10%EtOAc/hexanes) to give 32 mg of p-bromo benzoate 347 (0.054 mmol, 88%).347: [α]_(D)=+29.0 (c 0.78, CHCl₃); IR 2953, 2933, 1720, 1590, 1460,1273, 1037 cm⁻¹; ¹H NMR (400MHz, CDCl₃]) δ 7.90 (2H, d, J=8.8 Hz), 7.56(2H, d, J=8.8 Hz), 7.21 (1H, dd, J=7.2, 8.8 Hz), 7.07 (1H, d, J=8.8 Hz),6.81 (1H, d, J=7.2 Hz), 5.58-6.64 (1H, m), 5.50 (1H, br.dd, J=11.2, 15.2Hz), 5.36 (1H, br.dd, J=9.2, 15.2 Hz), 5.22 (1H, d, J=6.8 Hz), 5.14 (1H,d, J=6.8 Hz), 4.87 (1H, d, J=6.8 Hz), 4.81 (1H, d, J=6.8 Hz), 4.48-4.55(2H, m), 4.18 (1H, dd, J=3.2, 9.6 Hz), 3.72 (1H, dd, J=9.6, 16.4 Hz),3.43 (3H, s), 3.37 (3H, s), 3.34 (1H, br.d, J=16.4 Hz), 2.30-2.35 (1H,m), 2.05-2.19 (3H, m), 1.84 (1H, dd, J=8.4, 15.6 Hz), 1.72 (1H, ddd,J=11.6, 11.6, 14.0 Hz), 1.48 (1H, dd, J=9.6, 15.6 Hz), 0.87 (3H, d,J=7.2 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 168.3, 166.1, 154.5, 139.2, 131.9,131.8, 131.5, 131.3, 130.3, 129.4, 128.8, 128.3, 125.2, 124.0, 113.0,97.0, 94.6, 79.6, 71.4, 61.9, 56.2, 55.8, 38.0 (2 peaks), 35.8, 35.3,34.1, 13.5. HRMS Calcd for C₂₉H₃₅BrO₈Li (MLi⁺): 597.1675. Found:597.1682. 348: [α]_(D)=+16.2 (c 0.45, CHCl₃); IR 3380, 2920, 2847, 1717,1700, 1557, 1460, 1293, 1070 cm⁻¹; ¹H NMR (400 MHz, acetone[D₆]) δ 9.23(1H, s), 7.93 (2H, d (lr), J=8.8 (2.0) Hz), 7.68 (2H, d (lr), J=8.8(2.0) Hz), 7.16 (1H, dd, J=7.6, 8.0 Hz), 6.83 (1H, d, J=8.0 Hz), 6.71(1H, d, J=7.6 Hz), 5.63 (1H, ddd, J=4.8, 8.8, 8.8 Hz), 5.29-5.42 (2H,m), 4.50-4.58 (2H, m), 4.13 (1H, ddd, J=4.0, 4.4, 8.8 Hz), 3.62 (1H, dd,J=0.4, 4.8 Hz), 3.56 (1H, dd, J=7.2, 16.0 Hz), 3.40 (1H, br.d, J=16.0Hz), 2.25-2.32 (1H, m), 2.09-2.18 (2H, m), 1.86-1.94 (2H, m), 1.74-1.94(1H, m), 1.39 (1H, dd, J=8.8, 15.2 Hz), 0.87 (3H, d, J=6.8 Hz); ¹³C NMR(75 MHz, CDCl₃) δ 171.4, 166.1, 163.0, 142.4, 134.4, 133.2, 131.9,131.3, 129.0, 128.4, 126.5, 123.8, 117.0, 113.2, 72.3, 70.5, 61.8, 39.4,38.6, 37.5, 36.2, 34.6, 13.9; MS (EI) m/z (%): 506 (16), 505 (55), 504(45), 503 (50), 502 (32), 488 (18), 487 (40), 486 (58), 485 (100), 484(16). HRMS Calcd for C₂₅H₂₇BrO₆Li (ML⁺): 509.1151. Found: 509.1153.

[0314] To a solution of 336 (35 mg, 0.0556 mmol) in THF at 0° C. wasadded Bu₄NF (1 M in THF, 56 μL, 0.056 mmol). After stirring for 10 min,aq. NH₄Cl was added and the mixture was extracted with Et₂O. The organiclayer was washed with brine, dried (MgSO₄), filtered and concentrated.The residue was purified by FC (silicagel, 25% EtOAc in hexanes) to give24 mg of phenol xv (84%) as a colorless oil. xv: [α]_(D)=−8.0 (c 1.37,CHCl₃); IR 2955, 2929, 2856, 1725, 1652, 1463, 1451, 1361, 1293, 1249,1166, 1063, 972, 836 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 10.97 (1H, br.s,aryl-OH), 7.32 (1H, dd, J=7.5, 8.5 Hz), 7.00 (1H, ddd, J=7.5, 7.5, 15.6Hz), 6.90 (1H, dd, J=1.2, 8.4 Hz), 6.72 (1H, dd, J=1.2, 7.5 Hz),5.89-6.02 (2H, m), 5.42-5.52 (2H, m), 5.34 (1H, ddt, J=1.5, 1.5, 17.4Hz), 5.25 (1H, ddt, J=1.5, 1.5, 10.5 Hz), 5.02-5.14 (1H, m), 4.66 (2H,dt, J=1.5, 5.7 Hz), 3.70 (1H, dd, J=6.3, 16.2 Hz), 3.60 (1H, dd, J=3.0,8.4 Hz), 3.40 (1H, br.d, J=16.5 Hz), 2.58-2.64 (2H, m), 2.26-2.39 (1H,m), 2.01 (1H, dd, J=10.7, 15.0 Hz), 1.77-1.90 (2H, m), 1.38 (1H, ddd,J=1.2, 8.1, 15.0), 0.88 (9H, s), 0.87 (3H, d, J=6.9 Hz), 0.04 (3H, s),−0.03 (3H, s); ¹³C-NMR (100 MHz, CDCl₃) δ 170.8, 165.8, 162.8, 143.8,142.5, 134.2, 132.8, 132.4, 126.9, 124.4, 123.8, 118.4, 116.8, 113.4,73.8, 71.5, 65.3, 39.3, 38.5, 38.2, 37.5, 36.4, 26.0 (3C), 18.1, 14.0,−4.4, −4.7. HRMS Calcd for C₂₉H₄₂O₆SiLi (MLi⁺): 521.2911. Found:521.2912.

[0315] To a solution of phenol xv (23.5 mg, 0.0457 mmol) in CH₂Cl₂ (0.5mL) was added pyridine (42 μL), benzoyl chloride (31 μL, 0.262 mmol) at0° C. The mixture was stirred at 0° C. for 80 min, followed by theaddition of water (5 mL) and AcOH (150 μL). The resulting mixture wasextracted with ether (3×10 mL), dried over MgSO₄, filtered andconcentrated. Purification by FC (silicagel, 25% EtOAc in hexanes)provided 22 mg of benzoate 363 (78%) as a colorless oil. 363[α]_(D)=+4.0 (c 1.0, CHCl₃); IR 2928, 1745, 1726, 1452, 1268, 1228,1176, 1067, 836, 775, 707 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 8.16-8.20 (2H,m), 7.62-7.69 (1H, m), 7.49-7.55 (2H, m), 7.37 (1H, dd, J=7.8, 8.1 Hz),7.14 (1H, d, J=7.8 Hz), 7.11 (1H, d, J=8.1 Hz), 6.65 (1H, ddd, J=6.9,7.6, 15.6 Hz), 5.95 (1H, dddd, J=5.7, 5.7, 10.5, 17.1 Hz), 5.50 (1H,br.d, J=15.9 Hz), 5.30-5.50 (2H, m), 5.33 (1H, ddt, J=1.5, 1.5, 17.1Hz), 5.24 (1H, ddt, J=1.5, 1.5, 10.5 Hz), 5.14-5.22 (1H, m), 4.61 (2H,ddd, J=1.5, 1.5, 5.4 Hz), 4.23 (1H, dd, J=3.6, 8.7 Hz), 3.80 (1H, dd,J=9.3, 16.2 Hz), 3.41 (1H, br.d, J=16.2 Hz), 2.22-2.33 (1H, m), 2.08(1H, dddd, J=1.8, 6.9, 8.4, 15.0 Hz), 1.74-1.93 (2H, m), 1.58-1.73 (2H,m), 1.32 (1H, dd, J=8.7, 15.0 Hz), 0.88 (9H, s), 0.80 (3H, d, J=6.6 Hz),0.18 (3H, s), 0.12 (3H, s); ¹³C-NMR (75 MHz, CDCl₃) δ 166.6, 165.7,164.6, 148.2, 143.3, 140.3, 134.2, 132.5, 132.1, 130.4 (2C), 130.3,129.3, 129.0 (2C), 128.2, 128.1, 124.1, 121.4, 118.3, 73.4, 72.6, 65.2,38.2, 37.9, 37.4, 36.4, 26.1 (3C), 18.1, 13.2, −4.1, −4.2. HRMS Calcdfor C₃₆H₄₇O₇Si (MH⁺): 619.3091. Found: 619.3061.

[0316] To a solution of allyl ester 363 (20 mg, 0.0323 mmol) in THF (3mL) was added Pd(PPh₃)₄ (8 mg, 0.00646 mmol) and morpholine (28 μL,0.323 mmol). After 3 h at RT, the solvent was removed and the residuewas purified by FC (25% EtOAc in hexanes containing 5% AcOH). Carboxylicacid xvi (16 mg, 85%) was obtained as a colorless oil. xvi: [α]_(D)=+6.2(c 0.8, CHCl₃); IR 2928, 1746, 1699, 1452, 1269, 1228, 1068, 972, 775,707 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 8.18 (2H, dd, J=1.8, 7.5 Hz), 7.67(1H, app.t, J=7.5 Hz), 7.52 (2H, app.t, J=7.8 Hz), 7.38 (1H, app.t,J=8.1 Hz), 7.14 (1H, d, J=7.5 Hz), 7.13 (1H, d, J=7.5 Hz), 6.74 (1H,ddd, J=7.8, 8.7, 15.3 Hz), 5.28-5.50 (3H, m), 5.20 (1H, ddd, J=3.3, 8.1,11.1 Hz), 4.23 (1H, dd, J 3.6, 8.4 Hz), 3.81 (1H, dd, J=9.0, 16.2 Hz),3.41 (1H, br.d, J=16.5 Hz), 2.29 (1H, br.d, J=14.4 Hz), 2.02-2.12 (1H,m), 1.60-1.94 (4H, m), 1.31 (1H, dd, J=9.0, 15.3 Hz), 0.88 (9H, s), 0.80(3H, d, J=6.6 Hz), 0.19 (3H, s), 0.11 (3H, s); ¹³C-NMR (75 MHz, CDCl₃) δ170.7, 166.6, 164.6, 148.2, 145.8, 140.4, 134.3, 132.1, 130.4 (2C),130.3, 129.3, 129.0 (2C), 128.2, 128.0, 123.5, 121.5, 73.2, 72.7, 38.3,37.9, 37.3, 36.5, 26.0 (3C), 18.1, 13.6, −4.1, −4.2. HRMS Calcd forC₃₃H₄₃O₇Si (MH⁺): 579.2778. Found: 579.2776.

[0317] To a stirred solution of acid xvi (15.5 mg, 0.0268 mmol) and(PhO)₂P(O)N₃ (23 μL, 0.1071 mmol) in benzene (3 mL) was added Et₃N (19μL, 0.134 mmol) at RT. After stirring for 16 h at RT, the solvent wasremoved and the residue was purified by FC (17% EtOAc in hexanes). Theacyl azide xvii was obtained in 78% yield (12.5 mg). xvii: ¹H-NMR (400MHz, CDCl₃) δ 8.18 (2H, dd, J=1.2, 7.2 Hz), 7.67 (1H, m), 7.52 (2H,app.t, J=8.0 Hz), 7.38 (1H, app.t, J=8.0 Hz), 7.14 (1H, d, J=7.6 Hz),7.12 (1H, d, J=7.6 Hz), 6.72 (1H, ddd, J=6.8, 8.0, 14.8 Hz), 5.46 (1H,br.d, J=15.6 Hz), 5.44 (1H, dddd, J=2.4, 2.4, 10.8, 15.2 Hz), 5.33 (1H,dddd, J=2.0, 2.0, 9.2, 15.2 Hz), 5.19 (1H, ddd, J=3.2, 7.6, 7.6 Hz),4.23 (1H, dd, J=4.0, 9.2 Hz), 3.81(1H, dd, J=9.2, 16.4 Hz), 3.41(1H, br.d, J=16.4 Hz), 2.29 (1H, br.d, J=13.6 Hz), 2.01-2.09 (1H, m), 1.76-1.94(2H, m), 1.59-1.71 (2H, m), 1.25-1.31 (1H, m), 0.89 (9H, s), 0.80 (3H,d, J=6.4 Hz), 0.19 (3H, s), 0.12 (3H, s).

[0318] A solution of acylazide xvii (4 mg, 0.00663 mmol) and 1-pentanol(4.3 μL, 0.0398 mmol) in benzene (1 mL) was stirred for 6 h at 80° C.After removal of the solvent, the residue was treated with 370 μL of asolution of HF·pyridine in THF. After stirring for 4 days at RT, thereaction was quenched with a phosphate buffer (pH 7.0, 10 mL), extractedwith EtOAc (3×15 mL), dried over MgSO₄ and concentrated. Purification byFC (silicagel, 33% EtOAc in hexanes) gave 1.5 mg of compound 366 (41%).366: [α]_(D)=−48 (c 0.075, MeOH); IR 3439, 2960, 2929, 1713, 1678, 1452,1350, 1272, 1230, 974 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.14-8.18 (2H, m),7.72 (1H, apt.tt, J=1.6, 7.6 Hz), 7.58 (2H, app.t, J=7.6 Hz), 7.43 (1H,app.t, J=8.0 Hz), 7.19 (1H, d, J=8.0 Hz), 7.18 (1H, d, J=8.0 Hz), 6.14(1H, d, J=14.4 Hz), 5.44-5.51 (1H, m), 5.27-5.34 (1H, m), 5.12-5.18 (1H,m), 4.78 (1H, dd, J=7.6, 14.8), 4.18 (1H, dd, J=3.6, 9.2 Hz), 4.05 (2H,app.t, J=6.4 Hz), 3.72 (1H, dd, J=9.2, 16.0 Hz), 3.46 (1H, br.d, J=16.0Hz), 2.26 (1H, br), 1.71-1.90 (4H, m), 1.57-1.66 (3H, m), 1.26-1.38 (5H,m), 0.82 (3H, d, J=6.8 Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 168.5, 166.1,156.5, 149.5, 141.3, 135.6, 133.5, 131.3 (2C), 130.5, 130.2 (2C), 129.8,129.4, 129.3, 128.1, 126.6, 122.6, 105.2, 76.5, 72.2, 66.4, 39.0, 38.7,38.5, 37.2, 36.1, 29.9, 29.3, 23.5, 14.5, 13.6. MS (ES) m/z 550.27([M+H]⁺, 100).

[0319] HRMS Calcd for C₃₂H₃₉NO₇Li (MLi⁺): 556.2887. Found: 556.2871.

[0320] To a stirred solution of 336 (24 mg, 0.038 mmol) in THF (1.8 mL)was added dropwise concentrated HCl (55 μL) at RT. After stirring for 2h at RT, the reaction was quenched by the addition of saturated aq.NaHCO₃ (2 mL) and extracted with EtOAc (20 mL). The organic layer waswashed with brine, dried over MgSO₄, filtered and concentrated.Purification by FC (silicagel, 25% EtOAc in hexanes) provided 18 mg ofthe alcohol xviii (91%) as a colorless oil. xviii: [α]_(D)=+27 (c 0.9,CHCl₃); IR: 2930, 1724, 1581, 1457, 1280, 1167, 1115, 1066, 1028, 839cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.14 (1H, app.t, J=8.1 Hz), 6.98 (1H,ddt, J=7.5, 7.5, 15.6 Hz), 6.76 (1H, d, J=7.5 Hz), 6.74 (1H, d, J=8.1Hz), 5.89-6.30 (2H, m), 5.33-5.52 (3H, m), 5.34 (1H, app.qd, J=1.5, 17.4Hz), 5.26 (1H, app.qd, J=1.5, 10.5 Hz), 4.66 (2H, app.dt, J=1.5, 6.0Hz), 4.24 (1H, br.d, J=9.6 Hz), 3.66 (1H, dd, J=9.3, 16.2 Hz), 3.32 (1H,br.d, J=16.5 Hz), 2.55-2.72 (2H, m), 2.22-2.34 (1H, m), 1.80-1.95 (1H,m), 1.66-1.80 (2H, m), 1.44 (1H, dd, J=6.0, 15.6 Hz), 0.96 (9H, s), 0.87(3H, d, J=6.6 Hz), 0.21 (3H, s), 0.18 (3H, s); ¹³C-NMR (75 MHz, CDCl₃) δ168.4, 166.0, 153.0, 143.9, 139.0, 132.4, 131.5, 129.9, 128.8, 127.4,124.4, 123.4, 118.5, 118.0, 72.4, 71.4, 65.3, 38.5, 38.1, 38.0, 37.5,35.8, 26.0 (3C), 18.6, 13.9, −3.8, −3.9. HRMS Calcd for C₂₉H₄₂O₆SiLi(MLi⁺): 521.2911. Found: 521.2904.

[0321] To a solution of xviii (14 mg, 0.027 mmol) in pyridine (0.5 mL)was added benzoyl chloride (10 μL, 0.085 mmol) at 0° C. The reactionmixture was allowed to warm to RT and stirring was continued overnight.Aq. HCl (2N, 10 mL) was added and an extraction was performed with Et₂O(3×10 mL). The combined organic layers were washed with water and brine,dried (MgSO₄), filtered and concentrated. Purification by FC (silicagel,5% EtOAc in hexanes) provided 10 mg of the benzoate 364 (50%) as acolorless oil. 364: [α]_(D)=−20.8 (c 0.5, CHCl₃ ); IR 2930, 1722, 1457,1270, 1111, 1066, 839, 712; ¹H-NMR (400 MHz, CDCl₃) δ 8.07 (2H, dd,J=2.0, 7.2 Hz), 7.57 (1H, ddd, J=0.8, 7.2, 7.2 Hz), 7.45 (2H, app.t,J=8.0 Hz), 7.16 (1H, app.t, J=8.0 Hz), 7.00 (1H, ddd, J=7.2, 7.2, 15.6Hz), 6.78 (1H, d, J=7.6 Hz), 6.75 (1H, d, J=8.0 Hz), 5.90-6.2 (2H, m),5.64-5.74 (1H, m), 5.57-5.62 (1H, m), 5.36-5.45 (1H, m), 5.35 (1H, br.d,J=17.2 Hz), 5.26-5.33 (1H, m), 5.26 (1H, br.d, J=11.2 Hz), 4.67 (2H,br.d, J=6.0 Hz), 3.79 (1H, dd, J=9.6, 16.0 Hz), 3.34 (1H, br.d, J=16.0Hz), 2.60-2.70 (2H, m), 2.32-2.42 (2H, m), 1.78-1.92 (2H, m), 1.30 (1H,m), 0.94 (9H, s), 0.91 (3H, d, J=6.4 Hz), 0.18 (3H, s), 0.15 (3H, s);¹³C-NMR (75 MHz, CDCl₃) δ 167.7, 166.1, 165.9, 153.0, 143.8, 139.5,132.9, 132.4, 130.9, 130.8, 130.0, 129.9 (2C), 129.4, 128.6 (2C), 127.2,124.4, 123.4, 118.5, 118.0, 76.4, 71.4, 65.3, 38.2, 37.9, 33.3, 33.1,31.8, 26.0 (3C), 18.6, 14.3, −3.8, −3.9. HRMS Calcd for C₃₆H₄₆O₇SiLi(MLi⁺): 625.3173. Found: 625.3172.

[0322] Same procedure as described for the allyl ester deprotection ofcompound 363. Carboxylic acid xix: [α]_(D) ²³−14.2 (c 0.45, CHCl₃); IR2928, 1716, 1457, 1271, 1112, 838; ¹H-NMR (400 MHz, CDCl₃) δ 8.08 (2H,d, J 7.6 Hz), 7.54 (1H, app.t, J=7.2 Hz), 7.44 (2H, app.t, J=8.0 Hz),7.16 (1H, app.t, J=8.0 Hz), 7.09 (1H, ddd, J=7.2, 7.2, 15.6 Hz), 6.78(1H, d, J=7.6 Hz), 6.77 (1H, d, J=8.0 Hz), 6.01 (1H, d, J=15.6 Hz),5.66-5.75 (1H, m), 5.60 (1H, br.d, J=10.0 Hz), 5.42 (1H, br.dd, J=9.2,15.2 Hz), 5.29-5.35 (1H, m), 3.79 (1H, dd, J=8.8, 16.0 Hz), 3.35 (1H,br.d, J=16.0 Hz), 2.62-2.74 (2H, m), 2.33-2.44 (2H, m), 1.76-1.97 (3H,m), 0.94 (9H, s), 0.90 (3H, d, J=7.2 Hz), 0.19 (3H, s), 0.16 (3H, s);^(13 C-NMR ()75 MHz, CDCl₃) δ 170.7, 167.7, 166.1, 153.0, 146.2, 139.5,133.0, 130.82, 130.78, 130.0, 129.9 (2C), 129.4, 128.6 (2C), 127.1,123.9, 123.4, 118.0, 76.2, 71.2, 38.3, 37.9, 33.21, 32.18, 31.8, 26.0(3C), 18.6, 14.3, −3.8, −3.9. HRMS Calcd for C₃₃H₄₃O₇Si (MH⁺): 579.2778.Found: 579.2764.

[0323] Acyl azide xx was prepared according to the procedure describedfor the synthesis of acyl azide 336 in 96% yield. xx: ¹H-NMR (300 MHz,CDCl₃) δ 8.08 (2H, br.d, J=7.2 Hz), 7.59 (1H, br.t, J=7.5 Hz), 7.46 (2H,app.t, J=8.1 Hz), 7.16 (1H, app.t, J=7.8 Hz), 7.04 (1H, app.dt, J=6.9,15.9 Hz), 6.78 (1H, d, J=7.8 Hz), 6.75 (1H, d, J=8.4 Hz), 5.96 (1H,br.d, J=15.6 Hz), 5.62-5.74 (1H, m), 5.58 (1H, br.d, J=7.2 Hz), 5.40(1H, br.dd, J=9.0, 15.0 Hz), 5.26-5.34 (1H, m), 3.78 (1H, dd, J=9.6,16.2 Hz), 3.34 (1H, br.d, J=16.2 Hz), 2.58-2.74 (2H, m), 2.28-2.43 (2H,m), 1.75-1.96 (3H, m), 0.93 (9H, s), 0.90 (3H, d, J=6.3 Hz), 0.18 (3H,s), 0.14 (3H, s).

[0324] Compound 367 was prepared according to the procedure describedfor the preparation of compound 366 in 27% yield. 367: [α]_(D)=−17 (c0.12, MeOH); ¹H-NMR (400 MHz, CDCl₃) δ8.03 (2H, br.d, J=8.4 Hz), 7.60(1H, br.t, J=7.2 Hz), 7.48 (2H, app.t, J=8.0 Hz), 7.16 (1H, dd, J=7.6,8.4 Hz), 6.73 (1H, d, J=8.4 Hz), 6.70 (1H, d, J=7.6 Hz), 6.52 (1H, d,J=14.0 Hz), 5.53-5.63 (2H, m), 5.41 (1H, br.dd, J=7.6, 15.2 Hz),5.08-5.20 (2H, m), 4.07 (2H, app.t, J=6.8 Hz), 3.68 (1H, dd, J=8.4, 16.0Hz), 3.40 (1H, br.d, J=16.0 Hz), 2.22-2.44 (4H, m), 1.84-1.98 (3H, m),1.58-1.70 (2H, m), 1.30-1.40 (4H, m), 0.94 (3H, d, J=6.8 Hz), 0.93 (3H,t, J=7.0 Hz); MS (ES) m/z 572.27 ([M+Na]⁺, 18), 550.27 ([M+H]⁺, 100).HRMS Calcd for C₃₂H₃₉NO₇Li (MLi⁺): 556.2887. Found: 556.2881.

[0325] Compound xxi was prepared according to the procedure describedfor compound 337 in 95% yield. xxi: [α]_(D)=−94 (c 0.25, CHCl₃); IR2956, 1725, 1490, 1273; ¹H-NMR (300 MHz, CDCl₃) δ 7.18-7.30 (2H, m),6.80 (1H, d, J=8.4 Hz), 6.77 (1H, d, J=7.5 Hz), 5.92 (1H, d, J=15.6 Hz),5.30-5.56 (3H, m), 4.90 (1H, d, J=6.9 Hz), 4.82 (1H, d, J=6.9 Hz), 4.17(1H, dd, J=3.6, 9.3 Hz), 3.84 (3H, s), 3.71 (1H, dd, J=9.3, 16.5 Hz),3.45 (3H, s), 3.32 (1H, br.d, J=16.5 Hz), 2.62-2.74 (1H, m), 2.44-2.56(1H, m), 2.26-2.38 (1H, m), 1.63-1.83 (2H, m), 1.44 (1H, dd, J=9.0, 16.2Hz), 1.20-1.30 (1H, m), 0.87 (3H, d, J=6.6 Hz); ¹³C-NMR (75 MHz, CDCl₃)δ 171.6, 168.4, 157.0, 147.9, 139.2, 131.6, 130.4, 128.8, 124.3, 123.0,122.9, 109.5, 97.2, 79.6, 73.1, 55.9, 39.4, 37.9, 35.9, 34.3, 29.9,13.5. HRMS Calcd for C₂₃H₃₁O₇ (MH⁺): 419.2070. Found: 419.2055.

[0326] Acyl azide xxii was prepared according to the procedure describedfor acyl azide 338 in 68% yield. xxii: [α]_(D)=−112.4 (c 0.45, CHCl₃);IR 2926, 2141, 1726, 1696, 1584, 1468, 1273, 1174, 1084, 1040 cm⁻¹;¹H-NMR (300 MHz, CDCl₃) δ 7.25 (1H, app.t, J=7.8 Hz), 7.21 (1H, ddd,J=6.0, 8.7, 15.3 Hz), 6.83 (1H, d, J=8.1 Hz), 6.76 (1H, d, J=7.8 Hz),5.92 (1H, d, J=15.3 Hz), 5.28-5.58 (3H, m), 4.89 (1H, d, J=6.6 Hz), 4.81(1H, d, J=6.6 Hz), 4.15 (1H, dd, J=3.6, 9.3 Hz), 3.87 (3H, s), 3.71 (1H,dd, J=9.0, 16.5 Hz), 3.45 (3H, s), 3.32 (1H, br.d, J=16.5 Hz), 2.61-2.72(1H, m), 2.42-2.54 (1H, m), 2.26-2.38 (1H, m), 2.08-2.22 (1H, m),1.58-1.80 (1H, m), 1.42 (1H, dd, J=9.3, 14.7 Hz), 0.86 (3H, d, J=6.9Hz); ¹³C-NMR (75 MHz, CDCl₃) δ 171.8, 168.3, 156.9, 147.8, 139.3, 131.5,130.4, 128.8, 124.9, 124.3, 123.0, 109.6, 97.2, 79.6, 72.9, 55.97,55.87, 39.5, 37.93, 37.87, 35.9, 34.3, 13.5. HRMS Calcd for C₂₃H₂₉NO₆Li[MLi—N₂]⁺: 422.2155. Found: 422.2151.

[0327] A solution of the acyl azide xxii (8 mg, 0.01804 mmol) and1-pentanol (12 μL, 0.1082 mmol) in benzene (1 mL) was stirred for 7 h at80° C. After removal of the solvent, the residue was purified by flashchromatography (silicagel, 25% EtOAc in hexanes) to yield 5 mg ofcompound 365 (55%). 365: [α]_(D)=−70.8 (c 0.25, CHCl₃); IR 2926, 1723,1467, 1275, 1042 cm⁻¹; ¹³C-NMR (75 MHz, CDCl₃) δ 170.6, 158.4, 140.1,132.5, 131.4, 130.2, 127.9, 126.1, 123.7, 110.8, 106.7, 97.8, 80.7,76.2, 67.1, 66.4, 56.6, 56.0, 38.8, 38.6, 37.8, 35.7, 35.6, 30.0, 29.3,23.5, 14.5, 13.8. HRMS Calcd for C₂₈H₄₁NO₇Li (MLi⁺): 510.3043. Found:510.3049.

[0328] A solution of the acyl azide 338 (5 mg, 0.0081 mmol) and1-pentanethiol (6 μL, 0.0486 mmol) in benzene (1 mL) was stirred for 5 hat 80° C. After removal of the solvent, the residue was treated with 370μL of a solution HF-pyridine in THF (prepared by mixing 2 g commercialHF.Pyr, 10 mL pyridine and 27 mL THF). After stirring for 4 days at RT,the reaction was quenched with a phosphate buffer (pH 7.0; 10 mL),extracted with EtOAc (3×15 mL), dried over MgSO₄ and concentrated.Purification by FC (33% EtOAc in hexanes) gave 3 mg of compound 352(80%). 352: [α]_(D)=−20.0 (c 0.25, MeOH); IR 3208, 2928, 2855, 1663,1604, 1451, 1294, 1248, 1184, 948, 825 cm⁻¹; ¹H-NMR (300 MHz,acetone-d₆) δ 9.69 (1H, br.s, aryl-OH), 9.08 (1H, br.d, J=12.0 Hz, N—H),7.21 (1H, dd, J=7.5, 7.8 Hz), 6.83 (1H, dd, J=0.9, 7.8 Hz), 6.70-6.78(1H, m), 6.72 (1H, d, J=7.5 Hz), 5.22-5.40 (4H, m), 3.94 (1H, dd, J=3.9,9.0 Hz), 3.57 (1H, d, J=5.1 Hz), 3.46-3.52 (2H, m), 2.89 (2H, t, J=7.2Hz), 2.20-2.32 (1H, m), 1.74-1.94 (3H, m), 1.53-1.64 (2H, m), 1.28-1.38(5H, m), 0.88 (3H, t, J=6.9 Hz), 0.87 (3H, d, J=6.6 Hz); ¹³C-NMR (75MHz, CD₃OD) δ 171.2, 157.2, 140.8, 131.8, 130.9, 126.7, 123.2, 122.6,115.4, 108.6, 76.2, 72.1, 39.1, 38.9, 38.7, 37.4, 36.6, 32.1, 31.4,30.4, 23.4, 14.5, 13.7; MS (ES) m/z 484.33 ([M+Na]⁺, 8), 462.25 ([M+H]⁺,100). HRMS Calcd for C₂₅H₃₅NO₅SLi (MLi⁺): 468.2396. Found: 468.2400.

[0329] Compound 353 obtained from 338 in 53% yield. 353: [α]_(D)=−15.8(c 0.019, MeOH); IR 2928, 2853, 1724, 1453, 1376, 1293 cm⁻¹; ¹H-NMR (300MHz, CD₃COCD₃) δ 9.67 (1H, s, aryl-OH), 8.20 (1H, br.d, J=10.8 Hz, N—H),7.22 (1H, app.t, J=8.1 Hz), 6.83 (1H, dd, J=0.9, 8.1 Hz), 6.72 (1H,br.d, J=7.6 Hz), 6.54 (1H, dd, J=10.8, 14.4 Hz), 5.06-5.41 (7H, m), 4.57(2H, d, J=6.9 Hz), 3.93 (1H, ddd, J=3.6, 4.5, 8.1 Hz), 3.47-3.52 (2H,m), 2.22-2.42 (4H, m), 1.70-1.2.18 (10H, m), 1.71 (3H, s), 1.65 (3H, s),1.60 (3H, s), 1.59 (3H, s), 1.28-1.42 (1H, m), 0.87 (3H, d, J=6.6 Hz).MS (ES) m/z 580.37 ([M+H]⁺, 54). HRMS Calcd for C₃₅H₄₉NO₆Li (MLi⁺):586.3720. Found: 856.3690.

[0330] Acyl azide 338 (7 mg, 0.0114 mmol) in benzene (1 mL) was stirredat 80° C. for 6 h, after which the solvent was removed and the residuedissolved in THF. In a separate flask, a 0.19 M solution of1-lithio-phenylacetylene was prepared by deprotonation ofphenylacetylene (in THF) with ^(n)-BuLi (0.88 equiv.) at −78° C. To thissolution (0.2 mL, 0.19 M) in THF was added the isocyanate in THF (0.5mL) at −78° C. After stirring at −78° C. for 50 min, saturated NH₄Cl (3mL) and water (0.6 mL) were added and the resulting mixture was dilutedwith Et₂O (45 mL), the layers separated, and the organic layer washedwith brine, dried over MgSO₄, filtered and concentrated. Filtration oversilicagel (14% EtOAc in hexanes) provided a crude product (6 mg) whichwas treated with 370 μL of a solution HF pyridine in THF at RT. Afterstirring for 48 h at RT, the reaction was quenched with a phosphatebuffer (pH 7.0; 10 mL), extracted with EtOAc (3×15 mL), dried over MgSO₄and concentrated. Purification by FC (silicagel, 33% EtOAc in hexanes)gave 2 mg of compound 349 (56%). 349: [α]_(D)=−42 (c 0.1, MeOH ); IR2927, 1652, 1605, 1500, 1451, 1294, 1218, 1123; ¹H-NMR (400 MHz, CD₃OD)δ 7.58 (2H, app. dt, J=1.6, 6.8 Hz), 7.40-7.52 (3H, m), 7.14 (1H, app.t,J=8.0 Hz), 6.82 (1H, d, J=14.0 Hz), 6.74 (1H, d, J=8.4 Hz), 6.66 (1H, d,J=7.2 Hz), 5.51 (1H, ddd, J=7.6, 7.6, 14.4 Hz), 5.26-5.44 (3H, m), 4.14(1H, dd, J=3.6, 9.2 Hz), 3.58 (1H, dd, J=8.4, 16.4 Hz), 3.38 (1H, br),2.36-2.50 (2H, m), 2.29 (1H, br. d, J=14.4 Hz), 1.84-1.94(1H, m),1.72-1.84 (3H, m), 0.88 (3H, d, J=6.8 Hz); MS (ES) m/z (%): 460.22([M+H]⁺, 100) HRMS (FAB) Calcd for C28H29NO5Li (MLi⁺): 466.2206. Found:466:2204.

[0331] Compound 350 is prepared as described for the synthesis of 349(42% yield). 350: [α]_(D) ²³ −24.0 (c 0.1, MeOH ); IR 2929, 1653, 1498,1451, 1248, 1111; ¹H-NMR (400 MHz, CD₃OD) δ 7.13 (1H, dd, J=7.6, 8.0Hz), 6.75 (1H, d, J=10.4 Hz), 6.73 (1H, d, J=8.0 Hz), 6.66 (1H, d, J=7.6Hz), 5.43 (1H, ddd, J=7.2, 7.6, 14.0 Hz), 5.26-5.38 (3H, m), 4.12 (1H,dd, J=3.6, 8.8 Hz), 3.57 (1H, dd, J=8.8, 16.8 Hz), 3.37 (1H, m),2.24-2.44 (4H, m), 1.70-1.92 (3H, m), 1.32-1.62 (6H, m), 0.94 (3H, t,J=7.6 Hz), 0.87 (3H, d, J=6.8 Hz); MS (ES) m/z (%): 440.28 ([M+H]⁺,100), 462.21 ([M+Na]⁺, 10). HRMS (FAB) Calcd for C₂₆H₃₃NO₅Li (MLi⁺):446.2519. Found: 446.2513.

[0332] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of theinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A composition comprising a compound of formula:

wherein E is selected from the group consisting of:

X═O, S, NR²; Y═CH₂, O, S, NR²; Q=O, NH; F=ortho, meta, para substituentssuch as halogen, CN, OR², OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³,NR²SO₂R³, R³; R¹═H, Me; R²═R¹, straight chain saturated alkyl, straightchain unsaturated alkyl, branched chain alkyl, branched chainunsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl,CH₂heteroaryl, CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl,CHR¹CHR¹heterocycle; R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl,CR¹═CR¹heterocycle, C≡Caryl, C≡Cheteroaryl, C≡Cheterocycle; and Z is acontiguous linker whose presence completes an 11 to 15 membered ring.The linker can contain heteroatoms and substituents.
 2. A compositioncomprising a compound of formula:

wherein E is selected from the group consisting of:

X═O, S, NR² Y═CH₂, O, S, NR² Q=O, NH F=ortho, meta, para substituentssuch as halogen, CN, OR², OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³,NR²SO₂R³, R³ R¹═H, Me R²═R¹, straight chain saturated alkyl, straightchain unsaturated alkyl, branched chain alkyl, branched chainunsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl,CH₂heteroaryl, CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl,CHR¹CHR¹heterocycle R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl,CR¹═CR¹heterocycle, C≡Caryl, C≡Cheteroaryl, C≡Cheterocycle; and R⁴═R¹,C(O)R³, SO₂R³, R²
 3. A composition comprising a compound of formula:

wherein E is selected from the group consisting of:

X═O, S, NR² Y═CH₂, O, S, NR² Q=O, NH F=ortho, meta, para substituentssuch as halogen, CN, OR², OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³,NR²SO₂R³, R³ R¹═H, Me R²═R¹, straight chain saturated alkyl, straightchain unsaturated alkyl, branched chain alkyl, branched chainunsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH₂aryl,CH₂heteroaryl, CH₂heterocycle, CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl,CHR¹CHR¹heterocycle R³═R² or CR¹═CR¹aryl, CR¹═CR¹heteroaryl,CR¹═CR¹heterocycle, C≡Caryl, C≡Cheteroaryl, C≡Cheterocycle; and R⁴═R¹,C(O)R³, SO₂R³, R²
 4. A composition comprising a compound of formula:

where F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO₂R³, R³; R¹═H, Me; R²═R¹,straight chain saturated alkyl, straight chain unsaturated alkyl,branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl,aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl, CH₂heterocycle,CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle; R³═R² orCR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and R⁴═R¹, C(O)R³, SO₂R³, R²
 5. Acomposition comprising a compound is of formula:

where F=ortho, meta, para substituents such as halogen, CN, OR²,OC(O)R³, NO₂, OSO₂R³, NR²R², NR²C(O)R³, NR²SO³R³; R¹═H, Me; R²═R¹,straight chain saturated alkyl, straight chain unsaturated alkyl,branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl,aryl, heteroaryl, heterocycle, CH₂aryl, CH₂heteroaryl, CH₂heterocycle,CHR¹CHR¹aryl, CHR¹CHR¹heteroaryl, CHR¹CHR¹heterocycle; R³═R² orCR¹═CR¹aryl, CR¹═CR¹heteroaryl, CR¹═CR¹heterocycle, C≡Caryl,C≡Cheteroaryl, C≡Cheterocycle; and R⁴═R¹, C(O)R³, SO₂R³, R².
 6. Acomposition comprising a compound selected from the group consisting of:

wherein R=straight chain saturated alkyl or straight chain unsaturatedalkyl that is comprised of a chain of 5 to 8 carbons.
 7. A compositioncomprising a compound of formula:

wherein R=straight chain saturated alkyl or straight chain unsaturatedalkyl that is comprised of a chain of 5 to 8 carbons.
 8. A compositioncomprising a compound of formula:


9. A composition comprising a compound of formula:


10. A composition comprising a compound of formula:


11. A composition comprising a compound of formula:


12. A composition comprising a compound of formula:


13. A composition comprising a compound of formula:


14. A composition comprising a compound of formula:

where R═Bu; Ph.
 15. A composition comprising:

where R=Z,Z-hexadienyl; Z,E-hexadienyl; and a straight chain alkylcomprising 5-8 carbons.
 16. A composition comprising a compound offormula:


17. A composition comprising a compound of formula:


18. A composition comprising a compound of formula:


19. A composition comprising a compound of formula:


20. A composition comprising a compound of formula:


21. A composition comprising a compound of formula:


22. A composition comprising a compound of formula:


23. A composition comprising a compound of formula:


24. A composition comprising a compound of formula:


25. A composition comprising a compound of formula:


26. A composition comprising a compound of formula:


27. A composition comprising a compound of formula:


28. A composition comprising a compound of formula:

where R=a straight chain alkyl comprising 5-8 carbons, a straight chainalcohol, a straight chain diol, —CCBu, or —CCph.
 29. A compositioncomprising a compound is selected from the group consisting of:

wherein R=straight chain saturated alkyl or straight chain unsaturatedalkyl that is comprised of a chain of 5 to 8 carbons.
 30. A compositioncomprising a compound of formula:

wherein R=straight chain saturated alkyl or straight chain unsaturatedalkyl that is comprised of a chain of 5 to 8 carbons.
 31. A method oftreating or preventing osteoporosis, comprising the step ofadministering to a subject a therapeutically effective amount of thecompounds of any one of claims 1 through
 30. 32. A method for treatingcancer comprising the step of contacting a tumor cell within a subjectwith the compound of any one of claims 1 through 30 under conditionspermitting the uptake of the compound by the tumor cell.
 33. A method ofsuppressing growth of a tumor cell comprising contacting said cell witha compound of any one of claims 1 through 30, under conditionspermitting the uptake the compound by the tumor cell.
 34. A method ofinhibiting growth of proliferating cells comprising the step ofadministering, to the proliferating cells the compound of any one ofclaims 1 through 30 wherein the growth of the proliferating cells isinhibited.
 35. A process for preparing a salicylihalamide comprising thesteps of (a) synthesizing the compounds of formula:

(b) producing from the compounds of step (a), via an ring-closingmetathesis, the compound of formula:

wherein P=a hydroxyl protecting group.
 36. The method of claim 35further comprising modifying the compounds of step (a) to produce thefollowing compounds:

Wherein n=0, 1, 2, or 3 and m=1, 2, or 3; R=alkyl; and F=functionality;and (b) Producing from the compounds in step (a), as defined in step (b)of claim 48, the compounds of formula


37. A process for preparing a salicylihalamide comprising: (a)synthesizing the compound of formula:

wherein X═I, Br, Cl, OSO2Aryl; F=functionality as defined in claim 1;and P=a hydroxyl protecting group; (b) synthesizing the compound offormula:

(c) synthesizing from the compound of step (b), via a hydrometallation,the compound of formula:

wherein m=1, 2, or 3; R=alkyl; P=a hydroxyl protecting group and L_(n)Mis a ligated metal center with M=B, Zn, Zr, Pd, Cu, Li, Sn; and (d)producing from the compounds of step (a) and (c), via metal-catalyzedcross coupling, the compound of formula;


38. A process for preparing a salicylihalamide comprising the steps of:a) synthesizing a salicylihalamide benzolactone core; and b) adding aside chain to the salicylihalamide benzolactone core.
 39. A process forpreparing an apicularen comprising: a) synthesizing a apicularenbenzolactone core; and b) adding a chain to the apicularen benzolactonecore.